CN114716560B - Human papilloma virus 18 chimeric protein and application thereof - Google Patents

Human papilloma virus 18 chimeric protein and application thereof Download PDF

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CN114716560B
CN114716560B CN202110002251.3A CN202110002251A CN114716560B CN 114716560 B CN114716560 B CN 114716560B CN 202110002251 A CN202110002251 A CN 202110002251A CN 114716560 B CN114716560 B CN 114716560B
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CN114716560A (en
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许雪梅
张婷
王志荣
夏百成
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Institute of Basic Medical Sciences of CAMS
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Abstract

The invention relates to a human papillomavirus 18 type chimeric protein and application thereof. Specifically, the invention relates to a papillomavirus chimeric protein, which comprises HPV18 type L1 protein or a mutant of HPV18 type L1 protein and polypeptide from HPV59 type L2 protein inserted into the surface area of the HPV18 type L1 protein or the mutant of HPV18 type L1 protein, or consists of the HPV59 type L2 protein, wherein the amino acid sequence of the HPV18 type L1 protein is shown as SEQ ID NO.1, and the amino acid sequence of the HPV59 type L2 protein is shown as SEQ ID NO. 2.

Description

Human papilloma virus 18 chimeric protein and application thereof
Technical Field
The invention relates to the field of biotechnology, in particular to a human papillomavirus chimeric protein, a pentamer or virus-like particle formed by the human papillomavirus chimeric protein, and application of the human papillomavirus chimeric pentamer or human papillomavirus chimeric virus-like particle in preparing vaccines for preventing papillomavirus infection and diseases induced by the infection.
Background
Human papillomaviruses (human papillomavirus, HPV) are a class of non-enveloped small DNA viruses that infect epithelial tissues. Based on the homology of the major coat protein L1 amino acids of human papillomaviruses, more than 200 types of viruses have been identified, which are classified as alpha, beta, gamma, mu, eta. According to the infection site, it is classified into mucous membrane type and skin type. Mucosal HPV types are mainly infected in the genitourinary, perianal and oropharyngeal mucosal skin, are all of genus α, and are further classified into oncogenic HPV (oncogenic HPV) with transforming activity and low risk HPV (LR-HPV) which induces benign hyperplasia. Oncogenic HPV comprises 12 common high-risk types (including HPV types 16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, etc.), 1 possible high-risk type (HPV 68), and 10 more rare suspected high-risk types (HPV types 26, -30, -34, -53, -66, -67, -69, -70, -73, -82, -85, etc.). It was found that all the oncogenic HPV positive cancer tissues exhibited specific E6 x I mRNA expression, reduced expression of the oncogene Rb/P53 and cyclin CD1 and increased expression of p16.sup.ink 4a, indicating that the risk of cancer from infection with either oncogenic HPV is the same. There are about 12 low risk HPVs (HPV types 6, -7, -11, -13, -32, -40, -42, -43, -44, -54, -74, -91, etc.), where HPV types 6, -11 induce 90% of anal Zhou Shengshi device condyloma acuminatum and most recurrent papillomas of the respiratory tract in total. Cutaneous HPV types mainly infect skin tissues beyond the above sites, some of which (HPV 2, -27, -57) induce cutaneous wart, and others (HPV 5, -8, -38, etc.) are associated with the development of cutaneous squamous cell carcinoma and basal cell carcinoma.
The malignant tumors associated with oncogenic HPV infection are currently identified as follows: cervical cancer, vaginal cancer, labial cancer, penile cancer, perianal cancer, oropharyngeal cancer, tonsillar cancer, and oral cancer, with the greatest risk of cervical cancer. Cervical cancer is the third highest female malignancy worldwide, with a annual incidence of about 52.7 tens of thousands, with 28.5 tens of thousands in asia; the annual incidence of China is 7.5 ten thousand. The accumulation of 12 common high-risk HPVs induces 95.2% -96.5% of cervical cancers, and the accumulation of the rest 10 rare possible and suspicious high-risk HPVs induces about 3.29% of cervical cancers. HPV type 16 is a globally prevalent high-risk type with the highest detection rate in HPV-associated tumors such as cervical cancer, perianal cancer, penile cancer, vulvar cancer, etc., and precancerous lesions. The detection rates of HPV16 and HPV 18 in cervical cancer can reach 50-60% and 20% respectively. The accumulation of 12 common high-risk HPVs induces 95.2% -96.5% of cervical cancers, and the accumulation of the rest 10 rare possible and suspicious high-risk HPVs induces about 3.29% of cervical cancers.
HPV L1 virus-like particles (L1 VLPs) mainly induce specific neutralizing antibodies and protective responses, and the scope of vaccine protection can only be expanded by increasing the type of L1 VLPs. The 3 HPV vaccines on the market are L1 VLP vaccines, namely divalent vaccine (Cervarix, HPV 16/-18) of GSK, tetravalent vaccine (Gardasil, HPV 6/-11/-16/-18) of Merck and nine vaccine (Gardasil-9, HPV 6/-11/-16/-18/-31/-33/-45/-52/-58) respectively, wherein the nine vaccine with the widest protection range only covers the limited 7 high-risk types, 2 low-risk types (HPV 6/-11) and cannot prevent skin types. In addition, the L1 VLP vaccine cannot expand the protection range by limitlessly increasing the types of the L1 VLP, so that the L1 VLP vaccine is difficult to meet the prevention requirements of HPV infection related diseases.
Minor capsid proteins L2 of HPV are not immunologically active in the natural state, but the N-terminal polypeptides of L2 can induce cross-neutralizing antibodies and cross-protective reactions, with only weak immunogenicity, low titers of induced antibodies, and limited cross-neutralizing species of haplotype L2 antisera. A variety of conserved epitope peptides capable of inducing neutralizing antibodies are only found in 16L2N, wherein aa.17-38 is the main neutralizing epitope region thereof, and the monoclonal antibody RG-1 cross-neutralizing type recognizing the region is the most, so that the region is also called RG-1 epitope peptide, aa.21-31 is the core sequence of the neutralizing epitope, and the related research of RG-1 epitope peptide retains the homologous region of aa.21-31 no matter the sequence length.
The reported types of RG-1 used in vaccine studies are HPV type 4 RG-1, HPV type 6 RG-1, HPV type 16 RG-1, HPV type 17 RG-1, HPV type 31 RG-1, HPV type 33 RG-1, HPV type 45 RG-1, HPV type 51 RG-1, HPV type 58 RG-1, etc. [ C.Schellenbacher et al The Journal of investigative dermatology 2013,133 (12):2706-13；H.Seitz et al.,Vaccine 2014,32(22):2610-2617；B.Huber et al.,PLoS One 2015,10(3):e0120152；B.Huber et al., PLoS One 2017,12(1):e0169533；X.Chen et al.,Oncotarget 2017,8(38):63333-63344；X.Chen et al.,Human Vaccines&Immunotherapeutics 2018,14(8):2025-2033； PCT/CN2017/075402]modes used include VLP surface display, bacterial protein surface display (bacterial thioredoxin Trx, flagellin, cholera toxin mutant CRM 197), targeted igγr engineered antibodies and fusion in tandem of polymorphic L2 polypeptides containing RG-1 epitopes. However, studies have shown that the activity results of various RG-1 epitope peptide-related vaccines are poor, such as low titers of neutralizing antibodies to HPV16 induced by 3 cVLPs of HPV type 4 RG-1, HPV type 6 RG-1, HPV type 17 RG-1, cross-neutralization titers were undetectable [ B.Huber et al., PLoS One 2017,12 (1): e0169533; X.Chen et al, oncostarget 2017, 8 (38): 63333-63344 ]The method comprises the steps of carrying out a first treatment on the surface of the The 18cVLP displaying HPV45 type RG-1 induces very low titers of neutralizing antibodies to HPV18 (only 1/100 of the 18 type L1 VLP), and only cross-neutralizing oncogenic HPVs 45, 70 and 39, at very low titers, and at the highest only 100[B.Huber et al, PLoS One 2015, 10 (3): e 0120152)]The method comprises the steps of carrying out a first treatment on the surface of the Trx fusion protein antisera with surface displaying type 51 RG1 has narrow cross range, and the highest titer of cross neutralizing antibodies is only 500[H.Seitz et al, vaccine 2014,32 (22): 2610-2617]. In contrast, schellenbacher et al reported 16-type RG1-cVLP and 31-type RG1-cVLP, 33-type RG1-cVLP and 58-type RG1-cVLP reported previously by the inventor have better immunocompetence, and HPV16 neutralizing antibody titer induced by framework type VLP is as high as 10 5 (comparable to type 16L 1VLP induced), the corresponding RG-1 epitope induced L2 dependent cross-neutralizing antibodies have a broad neutralization range and relatively high titers (up to 6400) [ C.Schellebacher et al The Journal of investigative dermatology, 2013,133 (12): 2706-13; chen et al, oncostarget 2017,8 (38): 63333-63344; X.Chen et al, human Vaccines&Immunotherapeutics 2018,14(8):2025-2033； PCT/CN2017/075402]。
The above data suggest that there is a very large difference in immunogenicity of RG-1 epitope peptides derived from different HPV types. The inventors compared the immunogenicity of type 58 RG-1 and type 6 RG-1 in previous literature and found that type 58 RG-1 epitope peptide antisera cross-neutralized more (13 types), higher titers (up to 3200) and type 6 RG-1 epitope peptide antisera less neutralized (9 types), very low titers (up to 100 only) [ X.Chen et al, oncostarget 2017,8 (38): 63333-63344]. It is shown that although RG-1 epitope peptide regions have strong conservation among different types, the immunogenicity of RG-1 of different types is different, so that 1L 2 aa.17-36 homologous polypeptide is selected, and a chimeric protein vaccine is constructed, and the immune activity of the chimeric protein vaccine is unpredictable.
On the other hand, the study of HPV16 cVLP vaccine reported by Schellenbacher and Wang showed that, as well as inserting type 16 RG-1 epitope peptide into the surface region of type 16L 1 VLP vector, the immunological activity of the obtained various different type 16 RG 1-cVLPs has significant difference due to the difference of flanking sequence of type 16 RG-1 core epitope peptide sequence and insertion site and insertion mode, wherein, most preferably, cVLP of type 16 RG-1 is inserted into the DE loop region of type 16L 1, and worst, cVLP of type 16 RG-1 core sequence is inserted into the h4 region of type 16L 1. In addition, chen and box reported 33 type RG-1 cVLPs, but the vectors used were different, namely HPV 16L 1 VLPs and 18L1 VLPs, respectively, and although both reports selected the DE loop as an insertion site, the insertion region was 1 amino acid away, the epitope peptide length was 2 amino acids away, the activity difference of 33 type RG-1 dependent cross neutralizing antibodies induced by two 33 type RG 1-cVLPs obtained was quite remarkable, 33 type RG 1-cVLPs antisera could cross neutralize at least 12 types (with 2 types of titers > 1000), while 33 type RG1-18 cVLPs antisera only cross neutralize 7 types, with 6 types of neutralization titers (with 4 types of titers < 100) all being much lower than 33RG1-16 cVLPs antisera.
Thus, there is a need to develop a vaccine based on HPV L1 and HPV L2 chimeric proteins that is capable of producing high titer neutralizing antibodies against more HPV types of viruses.
Disclosure of Invention
In order to solve the technical problems, the inventor selects a plurality of 59 type RG-1 epitope peptides with different lengths for researching HPV cVLPs, and the result shows that the HPV18 type cVLPs obtained by the invention have strong immunogenicity, and the induced serum neutralizing antibodies can neutralize a plurality of types of HPVs of alpha 7 subgenera in a high titer.
Comparative analysis of the neutralizing Activity of 8 different types of RG-1 epitope immune serum in example 1 by the present inventors have unexpectedly found that immune serum of type 59 RG-1 epitope peptides cross-neutralize at least 17 types, particularly at titers of neutralizing HPV45, 59 and 16 types of 10 3 The activity of neutralizing alpha 7 is the best, and the cross neutralization activity of HPV16 is equivalent to the 16RG-1 with stronger immunogenicity.
Accordingly, the present invention is directed to a human papillomavirus chimeric protein for preparing a vaccine for preventing papillomavirus infection and infection-induced diseases.
The present invention is based on the unexpected findings of the inventors: insertion of an HPV type 59L 2 protein polypeptide into the surface region of a full-length or truncated HPV type 18L 1 protein increases the immunogenicity of the HPV type 59L 2 protein polypeptide, and the resulting chimeric protein can be expressed at high levels in e.coli or insect cell expression systems, can be assembled into VLPs, and can elicit broad-spectrum protective immune responses against multiple HPV types from different genera/subgenera. Relevant experimental results are provided in the examples herein.
In view of the above objects, the present invention provides, in one aspect, a human papillomavirus chimeric protein having a backbone of HPV 18L 1 protein or a mutant of HPV 18L 1 protein, said backbone having chimeric thereon at least one polypeptide derived from HPV type 59L 2 protein.
That is, in a first aspect of the present invention, there is provided a human papillomavirus chimeric protein comprising or consisting of an HPV18 type L1 protein or a mutant of HPV18 type L1 protein and a polypeptide from HPV59 type L2 protein inserted into the surface region of said HPV18 type L1 protein or mutant of HPV18 type L1 protein, wherein the amino acid sequence of said HPV18 type L1 protein is as shown in SEQ ID NO.1 and the amino acid sequence of said HPV59 type L2 protein is as shown in SEQ ID NO. 2.
In a preferred embodiment of the human papillomavirus chimeric protein according to the invention, said mutant of the HPV18 type L1 protein is the protein obtained by truncating 0-8 amino acids at the N-terminus and/or 0-32 amino acids at the C-terminus of said HPV18 type L1 protein.
In a preferred embodiment of the human papillomavirus chimeric protein according to the invention, the mutant of HPV type 18L 1 protein is selected from:
a mutant with 32 truncated amino acids at the C end of the amino acid sequence shown in SEQ ID No. 1;
A mutant (mut 1) in which amino acids 477, 478, 484, 496, 499, 504, 506 of the amino acid sequence shown in SEQ ID No.1 are substituted with glycine (G) and amino acids 485, 500, 502 are substituted with serine (S);
a mutant (mut 2) in which amino acids 477, 478, 485, 496, 499, 504, 506 of the amino acid sequence shown in SEQ ID No.1 are substituted with glycine (G) and amino acids 486, 500, 502 are substituted with serine (S);
a mutant (mut 3) in which amino acids 477, 478, 484, 496, 499, 502, 506 of the amino acid sequence shown in SEQ ID No.1 are substituted with glycine (G), amino acids 485, 500 are substituted with serine (S), and amino acid 504 is substituted with aspartic acid (D);
a mutant (mut 4) in which amino acids 477, 478, 485, 496, 502, 506 of the amino acid sequence shown in SEQ ID No.1 are substituted with glycine (G), amino acids 486, 500 are substituted with serine (S), and amino acids 499, 504 are substituted with aspartic acid (D);
a mutant (mut 5) in which amino acids 477, 484, 496, 499, 504, 506 of the amino acid sequence shown in SEQ ID No.1 are substituted with glycine (G) and amino acids 485, 500, 502 are substituted with serine (S); and
a mutant (mut 6) in which amino acids 477, 485, 496, 499, 504, 506 of the amino acid sequence shown in SEQ ID No.1 were replaced with glycine (G) and amino acids 486, 500, 502 were replaced with serine (S).
In a preferred embodiment of the human papillomavirus chimeric protein according to the invention, said polypeptide from the HPV type 59L 2 protein is selected from any consecutive 8-33 amino acid fragment within the 1-50 region of amino acids represented by SEQ ID No. 2; preferably, the polypeptide from HPV59 type L2 protein is HPV59 type L2 protein RG-1 epitope peptide or mutant epitope peptide thereof; further preferred, the polypeptide from HPV type 59L 2 protein consists of an amino acid sequence selected from the group consisting of: amino acids 17 to 32 shown in SEQ ID No.2, amino acids 16 to 35 shown in SEQ ID No.2, amino acids 17 to 37 shown in SEQ ID No.2, amino acids 16 to 37 shown in SEQ ID No.2, and sequences of 1 to 7 amino acids extended or truncated at the N-and/or C-terminus of the above amino acid sequences.
Most preferably, the amino acid sequence of the polypeptide from HPV59 type L2 protein is shown as SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 or SEQ ID No. 6.
Alternatively, the polypeptide from HPV59 type L2 protein is a polypeptide obtained by extension or truncation of 1-7 amino acids at the N-terminus and/or 1-7 amino acids at the C-terminus of the amino acid sequence shown in SEQ ID No. 3.
Alternatively, the polypeptide from HPV type 59L 2 protein may also be a polypeptide having greater than 60%, preferably greater than 70%, preferably greater than 80%, greater than 90%, even more preferably greater than 95% sequence identity to the amino acid sequence set forth in SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 or SEQ ID No. 6.
In a preferred embodiment of the human papillomavirus chimeric protein according to the invention, the HPV type 18L 1 protein may be derived from, for example, but not limited to, the L1 proteins from HPV18 variants in NCBI database, ATL15214.1, ATL14646.1, ARS43458.1, ARS43428.1, ARS43449.1, AGU90430.1, etc. Preferably, the amino acid sequence of the HPV18 type L1 protein is shown as SEQ ID No. 1.
In a preferred embodiment of the human papillomavirus chimeric protein of the invention, the polypeptide from HPV type 59L 2 protein is inserted into the surface region of HPV type 18L 1 protein or a mutant of HPV type 18L 1 protein; preferably, the DE loop or h4 region of said HPV18 type L1 protein or mutant of HPV18 type L1 protein is inserted; more preferably, said polypeptide from HPV59 type L2 protein is inserted between amino acids 134 and 135, between amino acids 137 and 138, between amino acids 432 and 433, or between amino acids 434 and 435 of said HPV18 type L1 protein or a mutant of said HPV18 type L1 protein by direct insertion, or between amino acids 121 to 124, or between amino acids 131 to 138, or between amino acids 431-433, or between amino acids 432-435 of said HPV18 type L1 protein or a mutant of said HPV18 type L1 protein by non-isometric substitution.
As used herein, the term "direct insertion" refers to insertion of a selected peptide fragment between two adjacent amino acids. For example, direct insertion between amino acids 134 and 135 of SEQ ID NO.1 refers to the insertion of a selected peptide fragment directly between amino acids 134 and 135 of SEQ ID NO. 1.
As used herein, the term "non-isometric substitution" refers to the insertion of a selected peptide fragment into a specified amino acid interval after deletion of the sequence of the specified amino acid interval. For example, a non-isometric substitution in the region of amino acids 121 to 124 of SEQ ID NO.1 refers to the insertion of a selected peptide fragment between amino acids 121 to 124 of SEQ ID NO.1 after deletion of amino acids 122-123 of SEQ ID NO. 1. Alternatively, in the direct insertion or non-isometric substitution mode, the polypeptide from HPV type 59L 2 protein comprises a 1 to 3 amino acid residue long linker at its N-and/or C-terminus.
Optionally, the linker is composed of any combination of amino acids selected from glycine (G), serine (S), alanine (a) and proline (P). Preferably, the N-terminal is a G (glycine) P (proline) linker and the C-terminal is a P (proline) linker.
In a preferred embodiment of the papillomavirus chimeric protein of the present invention, in the direct insertion mode, the amino acid sequence of the polypeptide derived from the HPV59 type L2 protein is SEQ ID No.4 or SEQ ID No.5, and the insertion site is between amino acid 137 and amino acid 138 of the HPV18 type L1 protein or a mutant of the HPV18 type L1 protein C-terminally truncated by 32 amino acids, and the obtained papillomavirus chimeric protein amino acid sequence is shown as SEQ ID No.7, SEQ ID No.8, SEQ ID No.9 or SEQ ID No. 10.
In a preferred embodiment of the human papillomavirus chimeric protein of the invention, in the direct insertion mode, the amino acid sequence of the polypeptide from HPV59 type L2 protein is SEQ ID No.3, the insertion site is between amino acids 432 and 433 or between amino acids 434 and 435 of the HPV18 type L1 protein or mutants of the HPV18 type L1 protein truncated by 32 amino acids at the C-terminus, and the obtained papillomavirus chimeric protein amino acid sequence is shown as SEQ ID No.11, SEQ ID No.12, SEQ ID No.13 or SEQ ID No. 14.
In a preferred embodiment of the human papillomavirus chimeric protein of the invention, in the direct insertion mode, the amino acid sequence of the polypeptide from the HPV59 type L2 protein is the sequence shown in SEQ ID No.4 or SEQ ID No.6 containing a GP linker at the N-terminal and a P linker at the C-terminal, and the insertion site is between amino acid 134 and amino acid 135 of the HPV18 type L1 protein or a mutant of the HPV18 type L1 protein truncated by 32 amino acids at the C-terminal, and the obtained papillomavirus chimeric protein amino acid sequence is shown in SEQ ID No.15, SEQ ID No.16, SEQ ID No.17 or SEQ ID No. 18.
In a preferred embodiment of the human papillomavirus chimeric protein of the invention, in the non-isometric substitution mode, after deleting the amino acid 132-137 region of the HPV18 type L1 protein or the mutant of HPV18 type L1 protein with 32 truncated amino acids at the C-terminus, a polypeptide from HPV59 type L2 protein is inserted between amino acids 131 and 138 of the HPV18 type L1 protein or the mutant of HPV18 type L1 protein with 32 truncated amino acids at the C-terminus, the polypeptide from HPV59 type L2 protein has an added glycine-proline linker at the N-terminus, the amino acid sequence of the polypeptide from HPV59 type L2 protein is shown as SEQ ID No.3, and the amino acid sequence of the obtained papillomavirus chimeric protein is shown as SEQ ID No.19 or SEQ ID No. 20.
In a preferred embodiment of the human papillomavirus chimeric protein of the invention, in said non-isometric substitution mode, after deletion of the amino acid 122-123 region of said HPV18 type L1 protein or of the mutant of said HPV18 type L1 protein truncated by 32 amino acids at the C-terminus, a polypeptide from HPV59 type L2 protein is inserted between amino acids 121 and 124 of said HPV18 type L1 protein or of the mutant of said HPV18 type L1 protein truncated by 32 amino acids at the C-terminus, in said direct insertion mode, the amino acid sequence of said polypeptide from HPV59 type L2 protein is shown in SEQ ID No.3, and the amino acid sequence of the obtained papillomavirus chimeric protein is shown in SEQ ID No.21 or SEQ ID No. 22.
In a preferred embodiment of the human papillomavirus chimeric protein of the invention, in said non-isometric substitution mode, after deleting amino acid 432 of said HPV18 type L1 protein or of said mutant of HPV18 type L1 protein C-terminally truncated by 32 amino acids, a polypeptide from HPV59 type L2 protein is inserted between amino acids 431 and 433 of said HPV18 type L1 protein or of said mutant of HPV18 type L1 protein C-terminally truncated by 32 amino acids, in said direct insertion mode, the amino acid sequence of said polypeptide from HPV59 type L2 protein is as shown in SEQ ID No.3, and the amino acid sequence of the obtained papillomavirus chimeric protein is as shown in SEQ ID No.23 or SEQ ID No. 24.
In a preferred embodiment of the human papillomavirus chimeric protein of the invention, in said non-isometric substitution mode, after deletion of amino acids 433-434 of said HPV18 type L1 protein or of said mutant of HPV18 type L1 protein truncated by 32 amino acids at the C-terminus, a polypeptide from HPV59 type L2 protein is inserted between amino acids 432 and 435 of said HPV18 type L1 protein or of said mutant of HPV18 type L1 protein truncated by 32 amino acids at the C-terminus, in said direct insertion mode the amino acid sequence of said polypeptide from HPV59 type L2 protein is as shown in SEQ ID No.3, and the amino acid sequence of the obtained papillomavirus chimeric protein is as shown in SEQ ID No.25 or SEQ ID No. 26.
In a preferred embodiment of the human papillomavirus chimeric protein of the invention, the polypeptide represented by SEQ ID No.4 is chimeric by direct insertion between amino acids 137 and 138 of said HPV18 type L1 protein mutant, said HPV18 type L1 protein mutant being selected from the group consisting of:
a mutant in which amino acids 477, 478, 484, 496, 499, 504, 506 of the amino acid sequence shown in SEQ ID No.1 are replaced with glycine (G) and amino acids 485, 500, 502 are replaced with serine (S), and the obtained papillomavirus chimeric protein has an amino acid sequence shown in SEQ ID No.27 (mut 1); or,
a mutant in which amino acids 477, 478, 485, 496, 499, 504, 506 of the amino acid sequence shown in SEQ ID No.1 are replaced with glycine (G) and amino acids 486, 500, 502 are replaced with serine (S), and the obtained papillomavirus chimeric protein has an amino acid sequence shown in SEQ ID No.28 (mut 2); or,
a mutant in which amino acids 477, 478, 484, 496, 499, 502, 506 of the amino acid sequence shown in SEQ ID No.1 are replaced with glycine (G), amino acids 485, 500 are replaced with serine (S) and amino acid 504 is replaced with aspartic acid (D), and the obtained papillomavirus chimeric protein has an amino acid sequence shown in SEQ ID No.29 (mut 3); or,
A mutant in which amino acids 477, 478, 485, 496, 502, 506 of the amino acid sequence shown in SEQ ID No.1 are replaced with glycine (G), amino acids 486, 500 are replaced with serine (S) and amino acids 499, 504 are replaced with aspartic acid (D), and the obtained papillomavirus chimeric protein has an amino acid sequence shown in SEQ ID No.30 (mut 4); or,
a mutant in which amino acids 477, 484, 496, 499, 504, 506 of the amino acid sequence shown in SEQ ID No.1 are replaced with glycine (G) and amino acids 485, 500, 502 are replaced with serine (S), and the obtained papillomavirus chimeric protein has an amino acid sequence shown in SEQ ID No.31 (mut 5); or,
a mutant in which amino acids 477, 485, 496, 499, 504, 506 of the amino acid sequence shown in SEQ ID No.1 were replaced with glycine (G) and amino acids 486, 500, 502 were replaced with serine (S) gave a papillomavirus chimeric protein having the amino acid sequence shown in SEQ ID No.32 (mut 6).
Another aspect of the invention relates to polynucleotides encoding the papillomavirus chimeric proteins described above.
The invention also provides a vector containing the polynucleotide and a cell containing the vector.
The polynucleotide sequence for encoding the papillomavirus chimeric protein is suitable for different expression systems. Alternatively, these nucleotide sequences are optimized by using E.coli codons for full gene expression in E.coli expression systems; or the insect cell codon is adopted for whole gene optimization, and the expression can be carried out at high level in an insect cell expression system.
The present invention also provides a polymer, preferably a papillomavirus chimeric pentamer or chimeric virus-like particle, comprising or formed from the papillomavirus chimeric protein described above.
The present invention also provides the use of the above papillomavirus chimeric proteins, papillomavirus chimeric pentamers or the above papillomavirus chimeric virus-like particles in the preparation of a vaccine for preventing papillomavirus infection and/or diseases induced by said papillomavirus infection, preferably, said papillomavirus infection-induced diseases including, but not limited to, cervical cancer, vaginal cancer, labial cancer, penile cancer, perianal cancer, oropharyngeal cancer, tonsil cancer and oral cancer;
preferably, the papillomavirus infection is one or more infections selected from the group consisting of: HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV70, HPV73; HPV6, HPV11, HPV2, HPV5, HPV27 and HPV57.
The present invention also provides a vaccine for preventing papillomavirus infection and infection-induced diseases comprising the above papillomavirus chimeric pentamer or chimeric virus-like particle, an adjuvant, and an excipient or carrier for the vaccine, preferably, a virus-like particle or chimeric virus-like particle further comprising at least one mucophilic group and/or dermatophilic group of HPV. Wherein the content of the virus-like particles is effective to induce protective immune response.
Optionally, the adjuvant is a human adjuvant.
Description and explanation of related terms in the invention
According to the present invention, the term "insect cell expression system" includes insect cells, recombinant baculoviruses, recombinant Bacmid and expression vectors. Wherein the insect cells are derived from commercially available cells, exemplified herein but not limited to: sf9, sf21, high Five.
According to the present invention, the term "prokaryotic expression system" includes, but is not limited to, E.coli expression systems. Wherein the expression host bacteria are derived from commercially available strains, exemplified herein but not limited to: BL21 (DE 3), BL21 (DE 3) plysS, C43 (DE 3), rosetta-gami B (DE 3).
According to the present invention, examples of the term "full length HPV type 18L 1 protein" include, but are not limited to, full length L1 proteins of equal length, the protein numbered ATL15070.1 in the NCBI database.
A gene fragment of a "truncated HPV18 type L1 protein" refers to a deletion of 1 or more amino acid-encoding nucleotides at its 5 'and/or 3' end compared to the wild type HPV18 type L1 protein gene, wherein the full length sequence of the "wild type HPV18 type L1 protein" is such as, but not limited to, the following sequences in the NCBI database: ATL15214.1, ATL14646.1, ARS43458.1, ARS43428.1, ARS43449.1, AGU90430.1, and the like.
According to the present invention, the term "vaccine excipient or carrier" refers to a compound selected from one or more of the group including, but not limited to: a pH adjustor, a surfactant, and an ion strength enhancer. For example, pH modifiers such as but not limited to phosphate buffers, surfactants including cationic, anionic, or nonionic surfactants such as but not limited to polysorbate 80 (Tween-80), and ionic strength enhancers such as but not limited to sodium chloride.
According to the present invention, the term "human adjuvant" refers to adjuvants that are clinically applicable to the human body, including various adjuvants that are currently approved and that may be approved in the future, such as, but not limited to, aluminum adjuvants, MF59, and various forms of adjuvant compositions.
According to the invention, the vaccine of the invention may take a patient acceptable form, including but not limited to oral or injection, preferably injection.
According to the invention, the vaccine of the invention is preferably used in unit dosage forms, wherein the dose of the protein virus-like particles in the unit dosage form is in the range of 5 μg to 100 μg, e.g. 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 μg, and ranges between any two of the above; preferably 30 μg to 60 μg.
Drawings
Fig. 1A-1B: expression identification of chimeric proteins in E.coli and insect cells in example 6 of the invention. The results showed that 26 chimeric proteins could be expressed in E.coli or insect cells, with 6 chimeric proteins expressed in both expression systems.
Fig. 1A: expression identification of chimeric proteins in E.coli: 1 is 18L1DE 137-138 59dES;2 is 18L1DE 137-138 59dE;3 is 18L1h4 432-433 59dE;4 is 18L1h4 434-435 59dE;5 is 18L1DE 134-135 59dES;6 is 18L1DE 134-135 59dE;7 is 18L1DE 131-138 59dE;8 is 18L1DE 121-124 59dE,9 is 18L1h4 431-433 59dE;10 is 18L1h4 432-435 59dE;11 is 18L1DE 137-138 59dES-mut1;12 is 18L1DE 137-138 59dES-mut2;13 is 18L1DE 137-138 59dES-mut3;14 is 18L1DE 137-138 59dES-mut4;15 is 18L1DE 137-138 59dES-mut5;16 is 18L1DE 137-138 /59dES-mut6；
Fig. 1B: expression of chimeric proteins in insect cells identification: 1 is 18L1 ΔCDE 137-138 59dES; 2. 18L1 ΔCDE 137-138 59dE;3 is 18L1 DeltaCh 4 432-433 59dE;4 is 18L1 DeltaCh 4 434-435 59dE;5 is 18L1 ΔCDE 134-135 59dES;6 is 18L1 ΔCDE 134-135 59dE;7 is 18L1 ΔCDE 131-138 59dE;8 is 18L1 ΔCDE 121-124 59dE,9 is 18L1 DeltaCh 4 431-433 59dE; 10. is 18L1 delta Ch4 432-435 59dE;11 is 18L1DE 137-138 59dES-mut1;12 is 18L1DE 137-138 59dES-mut2;13 is 18L1DE 137-138 59dES-mut3;14 is 18L1DE 137-138 59dES-mut4;15 is 18L1DE 137-138 59dES-mut5;16 is 18L1DE 137-138 /59dES-mut6。
Fig. 2A-2D: dynamic light scattering analysis results of cVLPs obtained after purification in example 6 of the present invention. The results show 18L1 ΔCDE 134-135 /59dE、18L1ΔCDE 134-135 /59dES、18L1ΔCDE 137-138 /59dE18L1 ΔCDE 137-138 The kinetic diameters of hydration of virus-like particles formed by the 59dES recombinant protein are 106.8nm, 113.3nm, 114.7nm and 122.9nm respectively, and the percentage of particle assembly is 100%.
Fig. 2A:18L1 ΔCDE 134-135 59dE; fig. 2B:18L1 ΔCDE 134-135 59dES; fig. 2C: 18L1 ΔCDE 137-138 59dE; fig. 2D:18L1 ΔCDE 137-138 /59dES。
Fig. 3A-3D: transmission electron microscopy observations of the cvlps obtained after purification in example 7 of the invention. A large number of virus-like particles are visible in the visual field, and the particles are uniform. cVLP is about 50nm in diameter, similar to the VLP of L1 protein in size. Bar=50 nm.
Fig. 3A:18L1 ΔCDE 134-135 59dE; fig. 3B:18L1 ΔCDE 134-135 59dES; fig. 3C: 18L1 ΔCDE 137-138 59dE; FIG. 3D.18L1ΔCDE 137-138 /59dES。
Fig. 4: the neutralizing activity of chimeric VLP mouse immune serum in example 10 of the invention against α7 subgeneric HPV pseudoviruses was tested. * : p <0.05.
Detailed Description
The invention will be further illustrated by the following non-limiting examples, which are well known to those skilled in the art, and many modifications can be made to the invention without departing from the spirit thereof, and such modifications also fall within the scope of the invention. The following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention as embodiments are necessarily varied. The terminology used in the description is for the purpose of describing particular embodiments only and is not intended to be limiting, the scope of the present invention being defined in the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Preferred methods and materials of the invention are described below, but any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention. The following experimental methods are all methods described in conventional methods or product specifications unless otherwise specified, and the experimental materials used are readily available from commercial companies unless otherwise specified. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and/or materials in the publications.
Example 1: immunocompetence detection of different types of RG-1 epitope peptides
RG-1 epitope peptides of HPV35, -39, -51, -53, -56, -59, -68, -82 were synthesized by chemical synthesis, the epitope peptide sequences are shown in Table 1, the polypeptides were synthesized by Shanghai Jier Biochemical Co., ltd, and each synthetic peptide was coupled to Keyhole Limpet Hemocyanin (KLH) after activating the carboxyl group by 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC, CAS No. 25952-53-8) in order to increase the immunogenicity of the synthetic peptide.
2.0-2.5kg body weight of New Zealand white rabbits are taken, randomly grouped, 2-4 of each group are injected into the back of the human body at multiple points for 4 days before immunization, 15mg of inactivated DH5a (PBS containing 0.5% v/v formaldehyde and treated for 24-48 hours at 37 ℃) which is fully mixed with an equal volume of Freund's complete adjuvant is injected into the back of the human body for immunization, and 1mg of KLH-polypeptide which is fully mixed with the equal volume of Freund's complete adjuvant is injected into the back of the human body and the inner side of the thigh at multiple points for the first immunization. The booster was performed 4 times, 2 weeks apart, with 0.5mg antigen and an equal volume of Freund's incomplete adjuvant (KLH-polypeptide mixed well. 2 weeks after the last immunization, blood was collected and serum was isolated.
The neutralizing antibody titer of the immune serum was measured using 17 HPV pseudoviruses, and the results are shown in table 2. The 59RG-1 epitope peptide has best immunological activity, and its antiserum can neutralize all 17 detection types, wherein the titer of neutralizing antibody of HPV45, -59, -16 is 10 3 The above, HPV5, -31, -18, -39, -68, -57 has a neutralizing antibody titer between 500-1000. Notably, 59RG-1 epitope peptide antisera had a high level of neutralizing antibodies to the 5 alpha 7-HPVs detected.
Methods for polypeptide synthesis, pseudovirus preparation and pseudovirus neutralization experiments are disclosed, for example, in patent CN 104418942a and 108676057a.
TABLE 1 sequence of synthesized different types of RG-1 epitope peptides
Identification device Synthetic peptide sequences SEQ ID NO.
HPV35 TQLYRTCKAAGTCPPDVIPKVEG 44
HPV39 STLYRTCKQSGTCPPDVVDKVEG 45
HPV51 TQLYSTCKAAGTCPPDVVNKVEG 46
HPV53 TQLYQTCKQSGTCPEDVINKIEH 47
HPV56 TQLYKTCKLSGTCPEDVVNKIEQ 48
HPV59 LYKTCKQ AGTCP SDVIN KVEGTT 49
HPV68 STLYKTCKQSGTCPPDVINKVEG 50
HPV82 TQLYSTCKAAGTCPPDVIPKVKG 51
TABLE 2 serum neutralizing antibody titres induced by different RG1-KLH conjugated peptides in rabbits
Example 2: synthesis of chimeric L1 protein gene and construction of expression vector
26 chimeric L1 proteins, respectively:
1) Chimeric L1 protein 18L1DE 137-138 59dES: the skeleton is full length HPV18 type L1 protein (sequence shown as SEQ ID No. 1), aa.17-32 polypeptide (shown as SEQ ID No. 4) of HPV59 type L2 protein is directly inserted into the DE ring aa.137/138 site of the protein, 18L1DE 137-138 The amino acid sequence of the 59dES chimeric protein is shown in SEQ ID No. 7. Coding 18L1DE 137-138 The polynucleotide sequence of/59 dES is constructed by adopting a complete gene synthesis mode through the optimal design of an escherichia coli codon;
2) Chimeric L1 protein 18L1DE 137-138 59dE: the skeleton is full length HPV18 type L1 protein (sequence shown as SEQ ID No. 1), aa.16-35 polypeptide (shown as SEQ ID No. 5) of HPV59 type L2 protein is directly inserted into the site of DE ring aa.137/138 of the protein, 18L1DE 137-138 The amino acid sequence of the 59dE chimeric protein is shown in SEQ ID No. 9. Coding 18L1DE 137-138 The polynucleotide sequence of 59dE is constructed by adopting a complete gene synthesis mode through the optimal design of an escherichia coli codon;
3) Chimeric L1 protein 18L1h4 432-433 59dE: the skeleton is full length HPV18 type L1 protein (sequence shown as SEQ ID No. 1), aa.17-37 polypeptide (shown as SEQ ID No. 3) of HPV59 type L2 protein is directly inserted into aa.432/433 site of h4 region, 18L1h4 432-433 59dE blockThe amino acid sequence of the synthetic protein is shown as SEQ ID No. 11. Coding 18L1h4 432-433 The polynucleotide sequence of 59dE is constructed by adopting a complete gene synthesis mode through the optimal design of an escherichia coli codon;
4) Chimeric L1 protein 18L1h4 434-435 59dE: the skeleton is full length HPV18 type L1 protein (sequence shown as SEQ ID No. 1), aa.17-37 polypeptide (shown as SEQ ID No. 3) of HPV59 type L2 protein is directly inserted into aa.434/435 site of h4 region, 18L1h4 434-435 The amino acid sequence of the 59dE chimeric protein is shown in SEQ ID No. 13. Coding 18L1h4 434-435 The polynucleotide sequence of 59dE is constructed by adopting a complete gene synthesis mode through the optimal design of an escherichia coli codon;
5) Chimeric L1 protein 18L1DE 134-135 59dES: the framework is full length HPV18 type L1 protein (the sequence is shown as SEQ ID No. 1), aa.17-32 polypeptide of HPV59 type L2 protein containing GP linker at N end and P linker at C end is directly inserted into the aa.134/135 site of DE ring (i.e. glycine-proline is added at N end and proline is added at C end of the sequence shown as SEQ ID No. 4), 18L1DE 134-135 The amino acid sequence of the 59dES chimeric protein is shown in SEQ ID No. 15. Coding 18L1DE 134-135 The polynucleotide sequence of/59 dES is constructed by adopting a complete gene synthesis mode through the optimal design of an escherichia coli codon;
6) Chimeric L1 protein 18L1DE 134-135 59dE: the framework is full length HPV18 type L1 protein (the sequence is shown as SEQ ID No. 1), aa.16-37 polypeptide of HPV59 type L2 protein containing GP linker at N end and P linker at C end is directly inserted into the aa.134/135 site of DE ring (i.e. glycine-proline is added at N end and proline is added at C end of the sequence shown as SEQ ID No. 6), 18L1DE 134-135 The amino acid sequence of the 59dE chimeric protein is shown in SEQ ID No. 17. Coding 18L1DE 134-135 The polynucleotide sequence of/59 dES is constructed by adopting a complete gene synthesis mode through the optimal design of an escherichia coli codon;
7) Chimeric L1 protein 18L1DE 131-138 59dE: the skeleton is full length HPV18 type L1 protein (sequence is shown as SEQ ID No. 1), aa.132-137 region is deleted, and HPV59 type L2 protein containing GP linker at N terminal is fused between aa.131/138aa.17-37 polypeptide (non-isometric substitution insertion in aa.132-137 region of HPV18 type L1 protein), glycine-proline and 18L1DE are added to the N-terminal of the insertion fragment with the amino acid sequence shown in SEQ ID No.3 131-138 The amino acid sequence of the 59dE chimeric protein is shown in SEQ ID No. 19. Coding 18L1DE 131-138 The polynucleotide sequence of 59dE is constructed by adopting a complete gene synthesis mode through the optimal design of an escherichia coli codon;
8) Chimeric L1 protein 18L1DE 121-124 59dE: the skeleton is full length HPV18 type L1 protein (the sequence is shown as SEQ ID No. 1), aa.122-123 region is deleted, aa.17-37 polypeptide of HPV59 type L2 protein is fused between aa.121/124 (non-equilong replacement insertion is performed in aa.122-133 region of HPV18 type L1 protein), the amino acid sequence of the insertion fragment is shown as SEQ ID No.3, 18L1DE 121-124 The amino acid sequence of the 59dE chimeric protein is shown as SEQ ID No. 21. Coding 18L1DE 121-124 The polynucleotide sequence of 59dE is constructed by adopting a complete gene synthesis mode through the optimal design of an escherichia coli codon;
9) Chimeric L1 protein 18L1h4 431-433 59dE: the skeleton is full length HPV18 type L1 protein (the sequence is shown as SEQ ID No. 1), aa.432 is deleted, aa.17-37 polypeptide of HPV59 type L2 protein is fused between aa.431/433 (non-equilong replacement insertion is performed in aa.431-433 region of HPV18 type L1 protein), the amino acid sequence of the insertion fragment is shown as SEQ ID No.3, 18L1h4 431-433 The amino acid sequence of the 59dE chimeric protein is shown in SEQ ID No. 23. Coding 18L1h4 431-433 The polynucleotide sequence of 59dE is constructed by adopting a complete gene synthesis mode through the optimal design of an escherichia coli codon;
10 Chimeric L1 protein 18L1h4 432-435 59dE: the skeleton is full length HPV18 type L1 protein (the sequence is shown as SEQ ID No. 1), aa.433-434 region is deleted, aa.17-37 polypeptide of HPV59 type L2 protein is fused between aa.432/435 (non-equilong replacement insertion is performed in aa.432-435 region of HPV18 type L1 protein), the amino acid sequence of the insertion fragment is shown as SEQ ID No.3, 18L1h4 432-435 The amino acid sequence of the 59dE chimeric protein is shown in SEQ ID No. 25. Coding 18L1h4 433-434 The polynucleotide sequence of 59dE is optimally set by the codon of the escherichia coliConstructing by adopting a total gene synthesis mode;
11 Chimeric L1 protein 18l1Δcde 137-138 59dES: HPV18 type L1 protein with skeleton of C-terminal truncated 32 amino acids (C-terminal truncated 32 amino acids of sequence SEQ ID No. 1), aa.17-32 polypeptide of HPV59 type L2 protein (shown as SEQ ID No. 4) and 18L1 delta CDE are directly inserted into aa.137/138 site of DE ring thereof 137-138 The amino acid sequence of the 59dES chimeric protein is shown in SEQ ID No. 8. Encoding 18L1ΔCDE 137-138 The polynucleotide sequence of/59 dES is constructed by adopting a complete gene synthesis mode through the optimized design of insect cell codons, and the nucleotide sequence is shown as SEQ ID No. 33;
12 Chimeric L1 protein 18l1Δcde 137-138 59dE: HPV18 type L1 protein with skeleton of C-terminal truncated 32 amino acids (C-terminal truncated 32 amino acids of sequence SEQ ID No. 1), aa.16-35 polypeptide (shown as SEQ ID No. 5) of HPV59 type L2 protein, 18L1 delta CDE, is directly inserted into aa.137/138 site of DE ring thereof 137-138 The amino acid sequence of the 59dE chimeric protein is shown in SEQ ID No. 10. Encoding 18L1ΔCDE 137-138 The polynucleotide sequence of/59 dE is constructed by adopting a total gene synthesis mode through the optimized design of insect cell codons, and the nucleotide sequence is shown as SEQ ID No. 34;
13 Chimeric L1 protein 18l1Δch4 432-433 59dE: HPV18 type L1 protein with skeleton of C-terminal truncated 32 amino acids (C-terminal truncated 32 amino acids of sequence SEQ ID No. 1), aa.17-37 polypeptide of HPV59 type L2 protein (shown as SEQ ID No. 3) and 18L1 delta Ch4 are directly inserted into aa.432/433 site of h4 region 432-433 The amino acid sequence of the 59dE chimeric protein is shown in SEQ ID No. 12. Encoding 18L1 DeltaCh 4 432-433 The polynucleotide sequence of/59 dE is constructed by adopting a total gene synthesis mode through the optimized design of insect cell codons;
14 Chimeric L1 protein 18l1Δch4 434-435 59dE: HPV18 type L1 protein with framework of C-terminal truncated 32 amino acids (C-terminal truncated 32 amino acids of sequence SEQ ID No. 1), aa.17-37 polypeptide (shown as SEQ ID No. 3) of HPV59 type L2 protein, 18L1 delta Ch4, is directly inserted at aa.434/435 site of h4 region 434-435 The amino acid sequence of the 59dE chimeric protein is shown as SEQ ID No.14Shown. Encoding 18L1 DeltaCh 4 434-435 The polynucleotide sequence of/59 dE is constructed by adopting a complete gene synthesis mode through the optimized design of insect cell codons;
15 Chimeric L1 protein 18l1Δcde 134-135 59dES: HPV18 type L1 protein with framework of C-terminal truncated 32 amino acids (C-terminal truncated 32 amino acids of sequence SEQ ID No. 1), aa.17-32 polypeptide of HPV59 type L2 protein with N-terminal GP linker and C-terminal P linker (i.e. glycine-proline is added at N-terminal and proline is added at C-terminal of sequence SEQ ID No. 4) and 18L1 delta CDE are directly inserted at aa.134/135 site of DE ring thereof 134-135 The amino acid sequence of the 59dES chimeric protein is shown as SEQ ID No. 16. Encoding 18L1ΔCDE 134-135 The polynucleotide sequence of/59 dES is constructed by adopting a complete gene synthesis mode through the optimized design of insect cell codons, and the nucleotide sequence is shown as SEQ ID No. 35;
16 Chimeric L1 protein 18l1Δcde 134-135 59dE: HPV18 type L1 protein with framework of C-terminal truncated 32 amino acids (C-terminal truncated 32 amino acids of sequence SEQ ID No. 1), aa.16-37 polypeptide of HPV59 type L2 protein with N-terminal GP linker and C-terminal P linker (i.e. glycine-proline is added at N-terminal and proline is added at C-terminal of sequence SEQ ID No. 6) and 18L1 delta CDE are directly inserted at aa.134/135 site of DE ring thereof 134-135 The amino acid sequence of the 59dE chimeric protein is shown as SEQ ID No. 18. Encoding 18L1ΔCDE 134-135 The polynucleotide sequence of/59 dE is constructed by adopting a total gene synthesis mode through the optimized design of insect cell codons, and the nucleotide sequence is shown as SEQ ID No. 36;
17 Chimeric L1 protein 18l1Δcde 131-138 59dE: HPV18 type L1 protein with framework of C-terminal truncated 32 amino acids (C-terminal truncated 32 amino acids of sequence SEQ ID No. 1), aa.132-137 region thereof is deleted, aa.17-37 polypeptide of HPV59 type L2 protein with N-terminal containing GP linker is fused between aa.131/138 (non-equilong replacement insertion in aa.132-137 region of HPV18 type L1 protein), glycine-proline is added to the N-terminal of the sequence shown in SEQ ID No.3 as the amino acid sequence of the insertion fragment, 18L1DE 131-138 The amino acid sequence of the 59dE chimeric protein is shown as SEQ ID No. 20. Coding 18L1DE 131-138 The polynucleotide sequence of/59 dE is constructed by adopting a total gene synthesis mode through the optimized design of insect cell codons, and the nucleotide sequence is shown as SEQ ID No. 37;
18 Chimeric L1 protein 18l1Δcde 121-124 59dE: HPV18 type L1 protein with framework of C-terminal truncated 32 amino acids (C-terminal truncated 32 amino acids of sequence SEQ ID No. 1), aa.122-123 region thereof is deleted, aa.17-37 polypeptide of HPV59 type L2 protein is fused between aa.121/124 (non-equilong substitution insertion in aa.122-133 region of HPV18 type L1 protein), the amino acid sequence of the insert is shown as SEQ ID No.3, 18L1 delta CDE 121-124 The amino acid sequence of the 59dE chimeric protein is shown in SEQ ID No. 22. Encoding 18L1ΔCDE 121-124 The polynucleotide sequence of/59 dE is constructed by adopting a total gene synthesis mode through the optimized design of insect cell codons;
19 Chimeric L1 protein 18l1Δch4 431-433 59dE: HPV18 type L1 protein with skeleton of C-terminal truncated 32 amino acids (C-terminal truncated 32 amino acids of sequence SEQ ID No. 1), aa.432 thereof is deleted, aa.17-37 polypeptide of HPV59 type L2 protein is fused between aa.431/433 (non-equilong substitution insertion in aa.431-433 region of HPV18 type L1 protein), the amino acid sequence of inserted fragment is shown as SEQ ID No.3, 18L1 delta Ch4 431-433 The amino acid sequence of the 59dE chimeric protein is shown in SEQ ID No. 24. Encoding 18L1 DeltaCh 4 431-433 The polynucleotide sequence of/59 dE is constructed by adopting a total gene synthesis mode through the optimized design of insect cell codons;
20 Chimeric L1 protein 18l1Δch4 432-435 59dE: HPV18 type L1 protein with framework of C-terminal truncated 32 amino acids (C-terminal truncated 32 amino acids of sequence SEQ ID No. 1), aa.433-434 region thereof is deleted, aa.17-37 polypeptide of HPV59 type L2 protein is fused between aa.432/435 (non-equilong substitution insertion in aa.432-435 region of HPV18 type L1 protein), the amino acid sequence of the insert fragment is shown as SEQ ID No.3, 18L1 delta Ch4 432-435 The amino acid sequence of the 59dE chimeric protein is shown in SEQ ID No. 26. Encoding 18L1 DeltaCh 4 432-435 The polynucleotide sequence of/59 dE is constructed by adopting a total gene synthesis mode through the optimized design of insect cell codons;
21 Embedding)L1 protein 18L1DE 137-138 59dES-mut1: mutant mut1 with skeleton of full length HPV18 type L1 protein (i.e. mutant with amino acids 477, 478, 484, 496, 499, 504, 506 replaced by glycine (G) and amino acids 485, 500, 502 replaced by serine (S)) has direct insertion of aa.17-32 polypeptide of HPV59 type L2 protein (as shown in SEQ ID No. 4) at the position aa.137/138 of DE loop, 18L1DE 137-138 The amino acid sequence of the 59dES-mut1 chimeric protein is shown in SEQ ID No. 27. Coding 18L1DE 137-138 The polynucleotide sequence of/59 dES-mut1 is constructed by adopting a complete gene synthesis mode through the optimal design of E.coli codons or the optimal design of insect cells codons, wherein 18L1DE of the optimal insect cell codons is adopted 137-138 The polynucleotide sequence of/59 dES-mut1 is shown in SEQ ID No. 38;
22 Chimeric L1 protein 18L1DE 137-138 59dES-mut2: mutant mut2 with skeleton of full length HPV18 type L1 protein (namely, amino acids 477, 478, 485, 496, 499, 504, 506 of sequence shown in SEQ ID No.1 are replaced by glycine and amino acids 486, 500, 502 are replaced by serine), aa.17-32 polypeptide of HPV59 type L2 protein (shown in SEQ ID No. 4) is directly inserted into the position aa.137/138 of DE loop of the mutant mut2, 18L1DE 137-138 The amino acid sequence of the 59dES-mut2 chimeric protein is shown in SEQ ID No. 28. Coding 18L1DE 137-138 The polynucleotide sequence of/59 dES-mut2 is constructed by adopting a complete gene synthesis mode through the optimal design of E.coli codons or the optimal design of insect cells codons, wherein 18L1DE of the optimal insect cell codons is adopted 137-138 The polynucleotide sequence of/59 dES-mut2 is shown in SEQ ID No. 39;
23 Chimeric L1 protein 18L1DE 137-138 59dES-mut3: mutant mut3 with full length HPV18 type L1 protein skeleton (namely, amino acids 477, 478, 484, 496, 499, 502 and 506 of SEQ ID No.1 are replaced by glycine, amino acids 485 and 500 are replaced by serine and amino acid 504 is replaced by aspartic acid), aa.17-32 polypeptide (shown as SEQ ID No. 4) of HPV59 type L2 protein is directly inserted into the site of DE loop aa.137/138 of the mutant mut3, 18L1DE 137-138 The amino acid sequence of the 59dES-mut3 chimeric protein is shown in SEQ ID No. 29.Coding 18L1DE 137-138 The polynucleotide sequence of/59 dES-mut3 is constructed by adopting a complete gene synthesis mode through the optimal design of E.coli codons or the optimal design of insect cells codons, wherein 18L1DE of the optimal insect cell codons is adopted 137-138 The polynucleotide sequence of/59 dES-mut3 is shown as SEQ ID No. 40;
24 Chimeric L1 protein 18L1DE 137-138 59dES-mut4: mutant mut4 with full length HPV18 type L1 protein skeleton (namely, amino acids 477, 478, 485, 496, 502 and 506 of SEQ ID No.1 are replaced by glycine, amino acids 486 and 500 are replaced by serine and amino acids 499 and 504 are replaced by aspartic acid), aa.17-32 polypeptide of HPV59 type L2 protein (shown as SEQ ID No. 4) is directly inserted into the site of DE loop aa.137/138 of the mutant mut4, 18L1DE 137-138 The amino acid sequence of the 59dES-mut4 chimeric protein is shown in SEQ ID No. 30. Coding 18L1DE 137-138 The polynucleotide sequence of/59 dES-mut4 is constructed by adopting a complete gene synthesis mode through the optimal design of E.coli codons or the optimal design of insect cells codons, wherein 18L1DE of the optimal insect cell codons is adopted 137-138 The polynucleotide sequence of/59 dES-mut4 is shown as SEQ ID No. 41;
25 Chimeric L1 protein 18L1DE 137-138 59dES-mut5: mutant mut5 with full length HPV18 type L1 protein skeleton (namely, amino acids 477, 484, 496, 499, 504, 506 of SEQ ID No.1 are replaced by glycine and amino acids 485, 500, 502 are replaced by serine), aa.17-32 polypeptide of HPV59 type L2 protein (shown as SEQ ID No. 4) is directly inserted into the position aa.137/138 of DE loop of the mutant mut5, 18L1DE 137-138 The amino acid sequence of the 59dES-mut5 chimeric protein is shown in SEQ ID No. 31. Coding 18L1DE 137-138 The polynucleotide sequence of/59 dES-mut5 is constructed by adopting a complete gene synthesis mode through the optimal design of E.coli codons or the optimal design of insect cells codons, wherein 18L1DE of the optimal insect cell codons is adopted 137-138 The polynucleotide sequence of/59 dES-mut5 is shown in SEQ ID No. 42;
26 Chimeric L1 protein 18L1DE 137-138 59dES-mut6: mutant mut6 with full length HPV18 type L1 protein as skeleton (i.e. amino group of sequence shown in SEQ ID No. 1)Acid 477, 485, 496, 499, 504, 506 is replaced by glycine and amino acid 486, 500, 502 is replaced by serine), aa.17-32 polypeptide (shown as SEQ ID No. 4) of HPV59 type L2 protein is directly inserted into the position aa.137/138 of DE loop, 18L1DE 137-138 The amino acid sequence of the 59dES-mut6 chimeric protein is shown in SEQ ID No. 32. Coding 18L1DE 137-138 The polynucleotide sequence of/59 dES-mut6 is constructed by adopting a complete gene synthesis mode through the optimal design of E.coli codons or the optimal design of insect cells codons, wherein 18L1DE of the optimal insect cell codons is adopted 137-138 The polynucleotide sequence of 59dES-mut5 is shown in SEQ ID No. 43.
The E.coli codon-optimized chimeric protein gene was digested with NdeI/XhoI, and then inserted into a commercial expression vector pET22b (manufactured by Novagen Co.). The insect cell codon-optimized chimeric protein gene was digested with BamHI/EcoRI and inserted into a commercial expression vector pFastBac1 (Invitrogen). The expression vectors containing the chimeric protein gene were obtained, and 16 expression vectors for E.coli were obtained, respectively: pET22b-18L1DE 137-138 /59dES，pET22b-18L1DE 137-138 /59dE， pET22b-18L1h4 432-433 /59dE，pET22b-18L1h4 434-435 /59dE， pET22b-18L1DE 134-135 /59dES，pET22b-18L1DE 134-135 /59dE， pET22b-18L1DE 131-138 /59dE，pET22b-18L1DE 121-124 /59dE， pET22b-18L1h4 431-433 /59dE，pET22b-18L1h4 432-435 /59dE， pET22b-18L1DE 137-138 /59dES-mut1，pET22b-18L1DE 137-138 /59dES-mut2， pET22b-18L1DE 137-138 /59dES-mut3，pET22b-18L1DE 137-138 /59dES-mut4， pET22b-18L1DE 137-138 /59dES-mut5，pET22b-18L1DE 137-138 59dES-mut6; the total of 16 expression vectors for insect refinement were: pFastBac1-18L 1. DELTA. CDE 137-138 /59dES， pFastBac1-18L1ΔCDE 137-138 /59dE，pFastBac1-18L1ΔCh4 432-433 /59dE， pFastBac1-18L1ΔCh4 434-435 /59dE，pFastBac1-18L1ΔCDE 134-135 /59dES， pFastBac1-18L1ΔCDE 134-135 /59dE，pFastBac1-18L1ΔCDE 131-138 /59dE， pFastBac1-18L1ΔCDE 121-124 /59dE，pFastBac1-18L1ΔCh4 431-433 /59dE， pFastBac1-18L1ΔCh4 432-435 /59dE，pFastBac1-18L1DE 137-138 /59dES-mut1， pFastBac1-18L1DE 137-138 /59dES-mut2，pFastBac1-18L1DE 137-138 /59dES-mut3， pFastBac1-18L1DE 137-138 /59dES-mut4，pFastBac1-18L1DE 137-138 /59dES-mut5， pFastBac1-18L1DE 137-138 59dES-mut6. The methods of cleavage, ligation and cloning are all well known, for example, from patent CN101293918B.
The amino acid sequence of the polypeptide used in the present invention is as follows:
HPV18 type L1 full-length amino acid
HPV59 type L2 full-length amino acid sequence
HPV59 type L2 aa.17-37
LYKTCKQAGTCPSDVINKVEG SEQ ID NO.3
HPV59 type L2 aa 17-32
LYKTCKQAGTCPSDVI SEQ ID NO.4
HPV59 type L2 aa.16-35
DLYKTCKQAGTCPSDVINKV SEQ ID NO.5
HPV59 type L2 aa.16-37
DLYKTCKQAGTCPSDVINKVEG SEQ ID NO.6
18L1DE 137-138 /59dES
18L1ΔCDE 137-138 /59dES
18L1DE 137-138 /59dE
18L1ΔCDE 137-138 /59dE
18L1h4 432-433 /59dE
18L1ΔCh4 432-433 /59dE
18L1h4 434-435 /59dE
18L1ΔCh4 434-435 /59dE
18L1DE 134-135 /59dES
18L1ΔCDE 134-135 /59dES
18L1DE 134-135 /59dE
18L1ΔCDE 134-135 /59dE
18L1DE 131-138 /59dE
18L1ΔCDE 131-138 /59dE
18L1DE 121-124 /59dE
18L1ΔCDE 121-124 /59dE
18L1h4 431-433 /59dE
18L1ΔCh4 431-433 /59dE
18L1h4 432-435 /59dE
18L1ΔCh4 432-435 /59dE
18L1DE 137-138 /59dES-mut1
18L1DE 137-138 /59dES-mut2
/>
18L1DE 137-138 /59dES-mut3
18L1DE 137-138 /59dES-mut4
18L1DE 137-138 /59dES-mut5
18L1DE 137-138 /59dES-mut6
The nucleotide sequence encoding the chimeric proteins of the invention is shown below:
18L1ΔCDE 137-138 /59dES nt
/>
18L1ΔCDE 137-138 /59dE nt
18L1ΔCDE 134-135 /59dES nt
/>
18L1ΔCDE 134-135 /59dE nt
/>
18L1ΔCDE 131-138 /59dE nt
/>
18L1DE 137-138 /59dES-mut1 nt
18L1DE 137-138 /59dES-mut2 nt
/>
18L1DE 137-138 /59dES-mut3 nt
18L1DE 137-138 /59dES-mut4 nt
18L1DE 137-138 /59dES-mut5 nt
/>
18L1DE 137-138 /59dES-mut6 nt
example 3: construction of recombinant Bacmid and recombinant baculovirus of chimeric L1 protein gene
Recombinant expression vectors pFastBac1-18L1ΔCDE comprising chimeric L1 gene were used, respectively 137-138 /59dES，pFastBac1-18L1ΔCDE 137-138 /59dE， pFastBac1-18L1ΔCh4 432-433 /59dE，pFastBac1-18L1ΔCh4 434-435 /59dE， pFastBac1-18L1ΔCDE 134-135 /59dES，pFastBac1-18L1ΔCDE 134-135 /59dE， pFastBac1-18L1ΔCDE 131-138 /59dE，pFastBac1-18L1ΔCDE 121-124 /59dE， pFastBac1-18L1ΔCh4 431-433 /59dE，pFastBac1-18L1ΔCh4 432-435 /59dE， pFastBac1-18L1DE 137-138 /59dES-mut1，pFastBac1-18L1DE 137-138 /59dES-mut2， pFastBac1-18L1DE 137-138 /59dES-mut3，pFastBac1-18L1DE 137-138 /59dES-mut4， pFastBac1-18L1DE 137-138 /59dES-mut5，pFastBac1-18L1DE 137-138 E.coli DH10Bac competence was transformed with/59 dES-mut6, recombinant Bacmid was obtained by screening, insect cells Sf9 were transfected with recombinant Bacmid, and recombinant baculovirus was amplified within Sf 9. Methods for screening recombinant Bacmid and amplifying recombinant baculoviruses are well known, for example, patent CN101148661B.
Example 4: expression of genes of chimeric L1 proteins in Sf9 cells
Sf9 cells were inoculated with 16 recombinant baculoviruses of chimeric L1 gene, expression of chimeric L1 protein was performed, fermentation broth was collected after culturing at 27 ℃ for about 88 hours, centrifugation was performed at 3000rpm for 15min, supernatant was discarded, and cells were washed with PBS for expression identification and purification. Methods of infection expression are disclosed, for example, in patent CN101148661B.
Example 5: expression of chimeric L1 protein genes in E.coli
Recombinant expression vectors pET22b-18L1DE containing chimeric L1 gene were used, respectively 137-138 /59dES， pET22b-18L1DE 137-138 /59dE，pET22b-18L1h4 432-433 /59dE， pET22b-18L1h4 434-435 /59dE，pET22b-18L1DE 134-135 /59dES， pET22b-18L1DE 134-135 /59dE，pET22b-18L1DE 131-138 /59dE， pET22b-18L1DE 121-124 /59dE，pET22b-18L1h4 431-433 /59dE， pET22b-18L1h4 432-435 /59dE，pET22b-18L1DE 137-138 /59dES-mut1， pET22b-18L1DE 137-138 /59dES-mut2，pET22b-18L1DE 137-138 /59dES-mut3， pET22b-18L1DE 137-138 /59dES-mut4，pET22b-18L1DE 137-138 /59dES-mut5， pET22b-18L1DE 137-138 E.coli BL21 (DE 3) was transformed with 59dES-mut 6.
The monoclonal was inoculated into 3ml of LB medium containing ampicillin and cultured overnight at 37 ℃. The bacterial liquid cultured overnight is prepared according to the following ratio of 1:100 is added into LB culture medium, cultured for 3 hours at 37 ℃, IPTG is added to the final concentration of 0.5 mu M when the OD600 reaches 0.8-1.0, and cultured for 12 hours at 16 ℃, and bacterial liquid is collected.
Example 6: expression identification of chimeric L1 proteins
Taking 1X 10 each of the cells expressing different chimeric L1 proteins described in example 4 and example 5 6 Separately, 10. Mu.l of each of the two samples was subjected to SDS-PAGE and Western blot identification by re-suspending the sample in 200. Mu.l of PBS, adding 50. Mu.l of 6×loading Buffer, and denaturing at 75℃for 8 minutes. As shown in FIGS. 1A-1B, 26 chimeric L1 proteins were expressed at high levels in insect cells or in prokaryotic expression systems, 18L1DE 137-138 /59dES，18L1DE 137-138 /59dE，18L1h4 432-433 /59dE， 18L1h4 434-435 /59dE，18L1DE 134-135 /59dES，18L1DE 134-135 /59dE， 18L1DE 131-138 /59dE，18L1DE 121-124 /59dE，18L1h4 431-433 /59dE， 18L1h4 432-435 /59dE，18L1DE 137-138 /59dES-mut1，18L1DE 137-138 /59dES-mut2， 18L1DE 137-138 /59dES-mut3，18L1DE 137-138 /59dES-mut4， 18L1DE 137-138 /59dES-mut5，18L1DE 137-138 59dES-mut6 is approximately 59kDa in size and the remaining 10 proteins are approximately 55kDa in size. Methods of SDS-PAGE electrophoresis and Western blot identification are disclosed, for example, in patent CN101148661B.
Example 7: comparison of expression level of chimeric L1 protein in insect cells
Taking the C-terminal truncated 32 amino acid 18L1 skeleton protein expression cells or chimeric L1 protein expression cells respectively taking C-terminal truncated 32 amino acid 18L1 as skeleton or 6 18L1 mutants as skeleton as described in example 4 6 And re-suspending in 200 μl PBS solution, and disrupting the cells by ultrasonic disruption (Ningbo Xinzhi ultrasonic disrupter, 2# probe, 100W, ultrasonic for 5s, interval 7s, total time 3 min), high speed centrifugation at 12000rpm for 10 min. The cleavage supernatant is collected and the L1 content of the supernatant is measured by a sandwich ELISA method, which is well known, for example, from patent CN104513826A.
Coating an ELISA plate with HPV18L1 monoclonal antibody prepared by the inventor, and incubating at 4 ℃ for overnight; the plates were blocked with 5% BSA-PBST for 2h at room temperature and washed 3 times with PBST. Lysates were serially diluted 2-fold with PBS and HPV18L1 VLP standard was also diluted in gradient, at a concentration from 2. Mu.g/ml to 0.0625. Mu.g/ml, and ELISA plates were added, 100. Mu.l per well, and incubated for 1h at 37 ℃. Plates were washed 3 times with PBST, add 1: HPV18L1 rabbit polyclonal antibody diluted at 3000 was incubated at 37℃for 1h at 100. Mu.l per well. Plates were washed 3 times with PBST, add 1:3000 dilution of HRP-labeled goat anti-mouse IgG (1:3000 dilution, china fir bridge Co.) was incubated at 37℃for 45 minutes. The plate was washed 5 times with PBST, 100. Mu.l of OPD substrate (Sigma Co.) was added to each well, color development was performed at 37℃for 5 minutes, the reaction was stopped with 50. Mu.l of 2M sulfuric acid, and the absorbance was measured at 490 nm. The concentration of HPV18L1 protein and 18L1 chimeric protein in the lysates was calculated according to a standard curve.
The results are shown in Table 3, 18L 1. DELTA.CDE of the present invention 134-135 /59dES、 18L1ΔCDE 137-138 /59dES、18L1ΔCh4 431-433 59dE and 18L1 DeltaCh 4 432-435 The expression level of/59 dE is very high and is equivalent to that of an HPV18L1 skeleton; in addition, chimeric proteins 18L1DE with C-terminal amino acid substitution of 18L1 mutant as backbone 137-138 /59dES-mut1、18L1DE 137-138 /59dES-mut4、 18L1DE 137-138 The expression level of/59 dES-mut5 is higher than that of HPV18L1 skeleton and the chimeric protein truncated at C-terminal.
TABLE 3 chimeric L1 protein expression level analysis
Example 8: purification of chimeric L1 proteins and dynamic light scattering particle size analysis
Taking a proper amount of cell fermentation broth of chimeric L1, re-suspending cells by using 10ml PBS, adding PMSF to a final concentration of 1mg/ml, performing ultrasonic disruption (Ningbo Xinzhi ultrasonic disrupter, 6# probe, 200W, ultrasonic treatment for 5s, interval of 7s and total time of 10 min), taking disruption supernatant, purifying, and performing the purification step at room temperature. VLPs were depolymerized by adding 4% beta-mercaptoethanol (w/w) to the lysate, and then the samples were filtered using a 0.22 μm filter, followed by DMAE anion exchange chromatography or CM cation exchange chromatography (20mM Tris,180mM NaCl,4% beta-ME, pH7.9 elution), TMAE anion exchange chromatography or Q cation exchange chromatography (20mM Tris,180mM NaCl,4% beta-ME, pH7.9 elution) and hydroxyapatite chromatography (100 mM NaH) 2 PO 4 30mM NaCl,4% beta-ME, pH 6.0 elution). The purified product was concentrated using a Planova ultrafiltration system and the buffer was replaced (20 mM NaH 2 PO 4 500mM NaCl, pH 6.0) facilitates VLP assembly. The above purification methods are disclosed, for example, in patent CN101293918B, CN1976718A and the like.
Chimeric protein pure product assembly processFound in (1) 18L1h4 431-433 /59dE、18L1h4 432-435 /59dE、 18L1ΔCh4 431-433 59dE and 18L1 DeltaCh 4 432-435 The 59dE was severely aggregated, and no aggregation was observed after assembly of the other chimeric proteins. The assembled chimeric protein solution was subjected to DLS particle size analysis (Zetasizer Nano ZS dynamic light scattering apparatus, malvern Co.) and the results are shown in Table 4, wherein 18L 1. DELTA.CDE 134-135 /59dE、 18L1ΔCDE 134-135 /59dES、18L1ΔCDE 137-138 /59dE、18L1ΔCDE 137-138 DLS analysis charts of/59 dES are shown in FIGS. 2A to 2D.
TABLE 4 chimeric L1 protein DLS analysis
Example 9: transmission electron microscopy of chimeric VLPs
The chimeric proteins were purified separately by the chromatographic purification method described in example 8, copper mesh was prepared using the assembled chimeric, stained with 1% uranium acetate, dried well and observed using JEM-1400 electron microscope (olympus). The results show that both E.coli and insect cell expressed chimeric proteins can be assembled into cVLPs with diameters of about 50 nm. Of which 18L 1. DELTA.CDE 134-135 /59dE、18L1ΔCDE 134-135 /59dES、 18L1ΔCDE 137-138 /59dE、18L1ΔCDE 137-138 The electron microscope pictures of/59 dES cVLP are shown in FIGS. 3A to 3D. Methods of copper mesh preparation and electron microscopy are disclosed, for example, in patent CN 101148661B.
Example 10: mouse immunization and neutralizing antibody titer assay for chimeric VLPs
BALB/c mice of 4-6 weeks of age were randomly grouped, 5 animals per group, combined with 10. Mu.g cVLP in combination with Al (OH) 3 50 μg and MPL adjuvant 5 μg immunized mice. Subcutaneous injections were performed and immunized 4 times at weeks 0,4,7, 10. Tail vein blood collection was carried out 2 weeks after the 4 th immunization, and serum was separated.
The results of the detection of neutralizing antibody titers in immune serum using 24 HPV pseudoviruses showed that the level and neutralization range of cross-neutralizing antibodies induced after immunization of mice with various crps produced by escherichia coli and insect cell expression systems were different. Wherein, as shown in Table 5, the insect cells expressed 18L 1. Delta. CDE 134-135 59dES and 18L1 ΔCDE 137-138 The 59dES cVLP antiserum can neutralize at least 23 pseudoviruses, 18L1ΔCDE 134-135 59dE and 18L1ΔCDE 137-138 The 59dE cVLP immune serum can neutralize at least 19 pseudoviruses. It is worth mentioning that 18L1 ΔCDE 134-135 59dES and 18L1 ΔCDE 137-138 59dES cVLP antiserum neutralizes all 6 detectable alpha 7-HPVs, particularly 18L 1. Delta. CDE 137-138 The antibody titer of the 59dES cVLP for cross-neutralizing alpha 7-HPV is more than 250, and the cVLP with the highest cross-neutralizing alpha 7-HPV capability is reported at present.
In addition, cVLP constructed by C-terminal truncated 32 amino acid 18L1 mutant in the invention can induce high-level neutralizing antibodies after mice are immunized by the strategy, wherein 18L1DE 137-138 Each neutralizing antibody induced by 59dES-mut4 was found to be equivalent in level to 18L 1. Delta. CDE 137-138 A equivalent of 59dES, notably 18L1DE 137-138 HPV39 and HPV59 neutralizing antibody titers of/59 dES-mut4 immune serum were both greater than 10 3 (as shown in Table 5 and FIG. 4).
Methods for pseudovirus preparation and pseudovirus neutralization experiments are disclosed, for example, in patent CN 104418942a.
TABLE 5 neutralizing antibody titres induced in mice by different cVLPs
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* ND means that no neutralizing antibodies were detected at the lowest dilution.
Sequence listing
<110> basic medical institute of the national academy of medical science
<120> a chimeric protein of human papillomavirus 18 and use thereof
<130> 300263CG
<160> 51
<170> SIPOSequenceListing 1.0
<210> 1
<211> 507
<212> PRT
<213> HPV 18
<400> 1
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Asn Val Ser Glu Asp Val Arg
130 135 140
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
145 150 155 160
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
165 170 175
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
180 185 190
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
195 200 205
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
210 215 220
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
225 230 235 240
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
245 250 255
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
260 265 270
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
275 280 285
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
290 295 300
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
305 310 315 320
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
325 330 335
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
340 345 350
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
355 360 365
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
370 375 380
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
385 390 395 400
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
405 410 415
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp
420 425 430
Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu Lys Phe Trp
435 440 445
Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp Gln Tyr Pro
450 455 460
Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu Arg Arg Lys Pro Thr
465 470 475 480
Ile Gly Pro Arg Lys Arg Ser Ala Pro Ser Ala Thr Thr Ser Ser Lys
485 490 495
Pro Ala Lys Arg Val Arg Val Arg Ala Arg Lys
500 505
<210> 2
<211> 464
<212> PRT
<213> HPV 59
<400> 2
Met Val Ser His Arg Ala Ala Arg Arg Lys Arg Ala Ser Ala Thr Asp
1 5 10 15
Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Ser Asp Val Ile
20 25 30
Asn Lys Val Glu Gly Thr Thr Leu Ala Asp Lys Ile Leu Gln Trp Thr
35 40 45
Ser Leu Gly Ile Phe Leu Gly Gly Leu Gly Ile Gly Thr Gly Ser Gly
50 55 60
Thr Gly Gly Arg Thr Gly Tyr Ile Pro Leu Gly Gly Arg Thr Asn Thr
65 70 75 80
Ile Val Asp Val Ser Pro Ala Lys Pro Pro Val Val Ile Glu Pro Val
85 90 95
Gly Pro Thr Asp Pro Ser Ile Val Thr Leu Val Glu Asp Ser Ser Val
100 105 110
Ile Thr Ser Gly Ala Pro Ala Pro Thr Phe Thr Gly Thr Ser Gly Phe
115 120 125
Glu Ile Ser Thr Ser Ser Thr Thr Thr Pro Ala Val Leu Asp Ile Thr
130 135 140
Pro Thr Ser Ser Val Gln Ile Ser Ser Ser Ser Phe Ile Asn Pro Ala
145 150 155 160
Phe Thr Asp Pro Ser Val Ile Glu Val Pro Gln Thr Gly Glu Ile Ser
165 170 175
Gly Asn Ile Leu Ile Ser Thr Pro Thr Ser Gly Ala His Gly Tyr Glu
180 185 190
Glu Ile Pro Met Gln Thr Phe Ala Thr Glu Gly Thr Gly Leu Glu Pro
195 200 205
Ile Ser Ser Thr Pro Asn Pro Thr Val Arg Arg Val Ala Gly Pro Arg
210 215 220
Leu Tyr Ser Arg Ala Asn Gln Gln Val Arg Val Ser Asp Ala Asn Phe
225 230 235 240
Leu Thr Arg Pro Ser Thr Phe Val Thr Tyr Asp Asn Pro Ala Tyr Asp
245 250 255
Pro Ile Asp Thr Thr Leu Thr Phe Asp Pro Ser Ser Glu Val Pro Asp
260 265 270
Pro Asp Phe Met Asp Ile Val Arg Leu His Arg Pro Ala Leu Thr Ser
275 280 285
Arg Arg Ser Thr Val Arg Phe Ser Arg Leu Gly Gln Arg Ala Thr Met
290 295 300
Phe Thr Arg Ser Gly Lys Gln Ile Gly Ala Arg Val His Phe Tyr His
305 310 315 320
Asp Ile Ser Pro Ile Pro His Ala Glu Asn Ile Glu Leu Gln Pro Leu
325 330 335
Val Ser Ser Gln Ala Ala Thr Asp Asp Ile Tyr Asp Ile Tyr Ala Asp
340 345 350
Ile Thr Asp Glu Ala Pro Thr Ser Thr Ala Asn Thr Ala Phe Thr Ile
355 360 365
Pro Lys Ser Ser Phe Gln Ser Leu Ser Leu Thr Arg Ser Ala Ser Ser
370 375 380
Thr Phe Ser Asn Val Thr Val Pro Leu Ala Thr Ala Trp Asp Val Pro
385 390 395 400
Val Asn Thr Gly Pro Asp Ile Val Leu Pro Asn Thr Asn Ile Val Gly
405 410 415
Pro Thr Tyr Ser Thr Thr Pro Phe Thr Thr Ile Gln Ser Ile Asn Ile
420 425 430
Glu Gly Thr Asn Tyr Phe Leu Trp Pro Ile Tyr Tyr Phe Leu Pro Arg
435 440 445
Lys Arg Lys Arg Val Pro Tyr Phe Phe Thr Asp Gly Ser Met Ala Phe
450 455 460
<210> 3
<211> 21
<212> PRT
<213> HPV 59
<400> 3
Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Ser Asp Val Ile
1 5 10 15
Asn Lys Val Glu Gly
20
<210> 4
<211> 16
<212> PRT
<213> HPV 59
<400> 4
Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Ser Asp Val Ile
1 5 10 15
<210> 5
<211> 20
<212> PRT
<213> HPV 59
<400> 5
Asp Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Ser Asp Val
1 5 10 15
Ile Asn Lys Val
20
<210> 6
<211> 22
<212> PRT
<213> HPV 59
<400> 6
Asp Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Ser Asp Val
1 5 10 15
Ile Asn Lys Val Glu Gly
20
<210> 7
<211> 523
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(523)
<223> 18L1DE137-138/59dES
<400> 7
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Leu Tyr Lys Thr Cys Lys Gln
130 135 140
Ala Gly Thr Cys Pro Ser Asp Val Ile Asn Val Ser Glu Asp Val Arg
145 150 155 160
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
165 170 175
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
180 185 190
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
195 200 205
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
210 215 220
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
225 230 235 240
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
245 250 255
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
260 265 270
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
275 280 285
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
290 295 300
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
305 310 315 320
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
325 330 335
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
340 345 350
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
355 360 365
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
370 375 380
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
385 390 395 400
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
405 410 415
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
420 425 430
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp
435 440 445
Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu Lys Phe Trp
450 455 460
Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp Gln Tyr Pro
465 470 475 480
Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu Arg Arg Lys Pro Thr
485 490 495
Ile Gly Pro Arg Lys Arg Ser Ala Pro Ser Ala Thr Thr Ser Ser Lys
500 505 510
Pro Ala Lys Arg Val Arg Val Arg Ala Arg Lys
515 520
<210> 8
<211> 491
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(491)
<223> 18L1ΔCDE137-138/59dES
<400> 8
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Leu Tyr Lys Thr Cys Lys Gln
130 135 140
Ala Gly Thr Cys Pro Ser Asp Val Ile Asn Val Ser Glu Asp Val Arg
145 150 155 160
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
165 170 175
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
180 185 190
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
195 200 205
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
210 215 220
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
225 230 235 240
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
245 250 255
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
260 265 270
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
275 280 285
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
290 295 300
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
305 310 315 320
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
325 330 335
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
340 345 350
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
355 360 365
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
370 375 380
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
385 390 395 400
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
405 410 415
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
420 425 430
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp
435 440 445
Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu Lys Phe Trp
450 455 460
Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp Gln Tyr Pro
465 470 475 480
Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu
485 490
<210> 9
<211> 527
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(527)
<223> 18L1DE137-138/59dE
<400> 9
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Asp Leu Tyr Lys Thr Cys Lys
130 135 140
Gln Ala Gly Thr Cys Pro Ser Asp Val Ile Asn Lys Val Asn Val Ser
145 150 155 160
Glu Asp Val Arg Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu
165 170 175
Cys Ile Leu Gly Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly
180 185 190
Thr Ala Cys Lys Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu
195 200 205
Glu Leu Lys Asn Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly
210 215 220
Tyr Gly Ala Met Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val
225 230 235 240
Pro Leu Asp Ile Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln
245 250 255
Met Ser Ala Asp Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg
260 265 270
Glu Gln Leu Phe Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly
275 280 285
Asp Thr Val Pro Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala
290 295 300
Ser Pro Gly Ser Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val
305 310 315 320
Thr Ser Asp Ser Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala
325 330 335
Gln Gly His Asn Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr
340 345 350
Val Val Asp Thr Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr
355 360 365
Gln Ser Pro Val Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr
370 375 380
Ser Arg His Val Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys
385 390 395 400
Thr Ile Thr Leu Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn
405 410 415
Ser Ser Ile Leu Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr
420 425 430
Thr Ser Leu Val Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr
435 440 445
Cys Gln Lys Asp Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys
450 455 460
Leu Lys Phe Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu
465 470 475 480
Asp Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu Arg
485 490 495
Arg Lys Pro Thr Ile Gly Pro Arg Lys Arg Ser Ala Pro Ser Ala Thr
500 505 510
Thr Ser Ser Lys Pro Ala Lys Arg Val Arg Val Arg Ala Arg Lys
515 520 525
<210> 10
<211> 495
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(495)
<223> 18L1ΔCDE137-138/59dE
<400> 10
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Asp Leu Tyr Lys Thr Cys Lys
130 135 140
Gln Ala Gly Thr Cys Pro Ser Asp Val Ile Asn Lys Val Asn Val Ser
145 150 155 160
Glu Asp Val Arg Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu
165 170 175
Cys Ile Leu Gly Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly
180 185 190
Thr Ala Cys Lys Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu
195 200 205
Glu Leu Lys Asn Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly
210 215 220
Tyr Gly Ala Met Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val
225 230 235 240
Pro Leu Asp Ile Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln
245 250 255
Met Ser Ala Asp Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg
260 265 270
Glu Gln Leu Phe Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly
275 280 285
Asp Thr Val Pro Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala
290 295 300
Ser Pro Gly Ser Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val
305 310 315 320
Thr Ser Asp Ser Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala
325 330 335
Gln Gly His Asn Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr
340 345 350
Val Val Asp Thr Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr
355 360 365
Gln Ser Pro Val Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr
370 375 380
Ser Arg His Val Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys
385 390 395 400
Thr Ile Thr Leu Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn
405 410 415
Ser Ser Ile Leu Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr
420 425 430
Thr Ser Leu Val Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr
435 440 445
Cys Gln Lys Asp Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys
450 455 460
Leu Lys Phe Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu
465 470 475 480
Asp Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu
485 490 495
<210> 11
<211> 528
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(528)
<223> 18L1h4432-433/59dE
<400> 11
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Asn Val Ser Glu Asp Val Arg
130 135 140
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
145 150 155 160
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
165 170 175
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
180 185 190
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
195 200 205
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
210 215 220
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
225 230 235 240
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
245 250 255
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
260 265 270
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
275 280 285
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
290 295 300
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
305 310 315 320
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
325 330 335
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
340 345 350
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
355 360 365
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
370 375 380
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
385 390 395 400
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
405 410 415
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp
420 425 430
Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Ser Asp Val Ile
435 440 445
Asn Lys Val Glu Gly Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp
450 455 460
Lys Leu Lys Phe Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp
465 470 475 480
Leu Asp Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu
485 490 495
Arg Arg Lys Pro Thr Ile Gly Pro Arg Lys Arg Ser Ala Pro Ser Ala
500 505 510
Thr Thr Ser Ser Lys Pro Ala Lys Arg Val Arg Val Arg Ala Arg Lys
515 520 525
<210> 12
<211> 496
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(496)
<223> 18L1ΔCh4432-433/59dE
<400> 12
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Asn Val Ser Glu Asp Val Arg
130 135 140
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
145 150 155 160
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
165 170 175
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
180 185 190
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
195 200 205
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
210 215 220
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
225 230 235 240
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
245 250 255
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
260 265 270
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
275 280 285
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
290 295 300
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
305 310 315 320
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
325 330 335
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
340 345 350
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
355 360 365
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
370 375 380
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
385 390 395 400
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
405 410 415
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp
420 425 430
Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Ser Asp Val Ile
435 440 445
Asn Lys Val Glu Gly Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp
450 455 460
Lys Leu Lys Phe Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp
465 470 475 480
Leu Asp Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu
485 490 495
<210> 13
<211> 528
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(528)
<223> 18L1h4434-435/59dE
<400> 13
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Asn Val Ser Glu Asp Val Arg
130 135 140
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
145 150 155 160
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
165 170 175
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
180 185 190
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
195 200 205
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
210 215 220
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
225 230 235 240
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
245 250 255
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
260 265 270
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
275 280 285
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
290 295 300
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
305 310 315 320
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
325 330 335
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
340 345 350
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
355 360 365
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
370 375 380
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
385 390 395 400
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
405 410 415
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp
420 425 430
Ala Ala Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Ser Asp
435 440 445
Val Ile Asn Lys Val Glu Gly Pro Ala Glu Asn Lys Asp Pro Tyr Asp
450 455 460
Lys Leu Lys Phe Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp
465 470 475 480
Leu Asp Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu
485 490 495
Arg Arg Lys Pro Thr Ile Gly Pro Arg Lys Arg Ser Ala Pro Ser Ala
500 505 510
Thr Thr Ser Ser Lys Pro Ala Lys Arg Val Arg Val Arg Ala Arg Lys
515 520 525
<210> 14
<211> 496
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(496)
<223> 18L1ΔCh4434-435/59dE
<400> 14
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Asn Val Ser Glu Asp Val Arg
130 135 140
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
145 150 155 160
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
165 170 175
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
180 185 190
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
195 200 205
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
210 215 220
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
225 230 235 240
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
245 250 255
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
260 265 270
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
275 280 285
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
290 295 300
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
305 310 315 320
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
325 330 335
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
340 345 350
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
355 360 365
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
370 375 380
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
385 390 395 400
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
405 410 415
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp
420 425 430
Ala Ala Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Ser Asp
435 440 445
Val Ile Asn Lys Val Glu Gly Pro Ala Glu Asn Lys Asp Pro Tyr Asp
450 455 460
Lys Leu Lys Phe Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp
465 470 475 480
Leu Asp Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu
485 490 495
<210> 15
<211> 526
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(526)
<223> 18L1DE134-135/59dES
<400> 15
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Gly Pro Leu Tyr Lys Thr Cys Lys Gln Ala
130 135 140
Gly Thr Cys Pro Ser Asp Val Ile Pro Ala Thr Ser Asn Val Ser Glu
145 150 155 160
Asp Val Arg Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys
165 170 175
Ile Leu Gly Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr
180 185 190
Ala Cys Lys Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu
195 200 205
Leu Lys Asn Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr
210 215 220
Gly Ala Met Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro
225 230 235 240
Leu Asp Ile Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met
245 250 255
Ser Ala Asp Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu
260 265 270
Gln Leu Phe Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp
275 280 285
Thr Val Pro Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser
290 295 300
Pro Gly Ser Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr
305 310 315 320
Ser Asp Ser Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln
325 330 335
Gly His Asn Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val
340 345 350
Val Asp Thr Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln
355 360 365
Ser Pro Val Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser
370 375 380
Arg His Val Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr
385 390 395 400
Ile Thr Leu Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser
405 410 415
Ser Ile Leu Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr
420 425 430
Ser Leu Val Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys
435 440 445
Gln Lys Asp Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu
450 455 460
Lys Phe Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp
465 470 475 480
Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu Arg Arg
485 490 495
Lys Pro Thr Ile Gly Pro Arg Lys Arg Ser Ala Pro Ser Ala Thr Thr
500 505 510
Ser Ser Lys Pro Ala Lys Arg Val Arg Val Arg Ala Arg Lys
515 520 525
<210> 16
<211> 494
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(494)
<223> 18L1ΔCDE134-135/59dES
<400> 16
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Gly Pro Leu Tyr Lys Thr Cys Lys Gln Ala
130 135 140
Gly Thr Cys Pro Ser Asp Val Ile Pro Ala Thr Ser Asn Val Ser Glu
145 150 155 160
Asp Val Arg Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys
165 170 175
Ile Leu Gly Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr
180 185 190
Ala Cys Lys Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu
195 200 205
Leu Lys Asn Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr
210 215 220
Gly Ala Met Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro
225 230 235 240
Leu Asp Ile Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met
245 250 255
Ser Ala Asp Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu
260 265 270
Gln Leu Phe Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp
275 280 285
Thr Val Pro Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser
290 295 300
Pro Gly Ser Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr
305 310 315 320
Ser Asp Ser Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln
325 330 335
Gly His Asn Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val
340 345 350
Val Asp Thr Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln
355 360 365
Ser Pro Val Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser
370 375 380
Arg His Val Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr
385 390 395 400
Ile Thr Leu Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser
405 410 415
Ser Ile Leu Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr
420 425 430
Ser Leu Val Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys
435 440 445
Gln Lys Asp Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu
450 455 460
Lys Phe Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp
465 470 475 480
Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu
485 490
<210> 17
<211> 532
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(532)
<223> 18L1DE134-135/59dE
<400> 17
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Gly Pro Asp Leu Tyr Lys Thr Cys Lys Gln
130 135 140
Ala Gly Thr Cys Pro Ser Asp Val Ile Asn Lys Val Glu Gly Pro Ala
145 150 155 160
Thr Ser Asn Val Ser Glu Asp Val Arg Asp Asn Val Ser Val Asp Tyr
165 170 175
Lys Gln Thr Gln Leu Cys Ile Leu Gly Cys Ala Pro Ala Ile Gly Glu
180 185 190
His Trp Ala Lys Gly Thr Ala Cys Lys Ser Arg Pro Leu Ser Gln Gly
195 200 205
Asp Cys Pro Pro Leu Glu Leu Lys Asn Thr Val Leu Glu Asp Gly Asp
210 215 220
Met Val Asp Thr Gly Tyr Gly Ala Met Asp Phe Ser Thr Leu Gln Asp
225 230 235 240
Thr Lys Cys Glu Val Pro Leu Asp Ile Cys Gln Ser Ile Cys Lys Tyr
245 250 255
Pro Asp Tyr Leu Gln Met Ser Ala Asp Pro Tyr Gly Asp Ser Met Phe
260 265 270
Phe Cys Leu Arg Arg Glu Gln Leu Phe Ala Arg His Phe Trp Asn Arg
275 280 285
Ala Gly Thr Met Gly Asp Thr Val Pro Gln Ser Leu Tyr Ile Lys Gly
290 295 300
Thr Gly Met Arg Ala Ser Pro Gly Ser Cys Val Tyr Ser Pro Ser Pro
305 310 315 320
Ser Gly Ser Ile Val Thr Ser Asp Ser Gln Leu Phe Asn Lys Pro Tyr
325 330 335
Trp Leu His Lys Ala Gln Gly His Asn Asn Gly Val Cys Trp His Asn
340 345 350
Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser Thr Asn Leu Thr
355 360 365
Ile Cys Ala Ser Thr Gln Ser Pro Val Pro Gly Gln Tyr Asp Ala Thr
370 375 380
Lys Phe Lys Gln Tyr Ser Arg His Val Glu Glu Tyr Asp Leu Gln Phe
385 390 395 400
Ile Phe Gln Leu Cys Thr Ile Thr Leu Thr Ala Asp Val Met Ser Tyr
405 410 415
Ile His Ser Met Asn Ser Ser Ile Leu Glu Asp Trp Asn Phe Gly Val
420 425 430
Pro Pro Pro Pro Thr Thr Ser Leu Val Asp Thr Tyr Arg Phe Val Gln
435 440 445
Ser Val Ala Ile Thr Cys Gln Lys Asp Ala Ala Pro Ala Glu Asn Lys
450 455 460
Asp Pro Tyr Asp Lys Leu Lys Phe Trp Asn Val Asp Leu Lys Glu Lys
465 470 475 480
Phe Ser Leu Asp Leu Asp Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val
485 490 495
Gln Ala Gly Leu Arg Arg Lys Pro Thr Ile Gly Pro Arg Lys Arg Ser
500 505 510
Ala Pro Ser Ala Thr Thr Ser Ser Lys Pro Ala Lys Arg Val Arg Val
515 520 525
Arg Ala Arg Lys
530
<210> 18
<211> 500
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(500)
<223> 18L1ΔCDE134-135/59dE
<400> 18
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Gly Pro Asp Leu Tyr Lys Thr Cys Lys Gln
130 135 140
Ala Gly Thr Cys Pro Ser Asp Val Ile Asn Lys Val Glu Gly Pro Ala
145 150 155 160
Thr Ser Asn Val Ser Glu Asp Val Arg Asp Asn Val Ser Val Asp Tyr
165 170 175
Lys Gln Thr Gln Leu Cys Ile Leu Gly Cys Ala Pro Ala Ile Gly Glu
180 185 190
His Trp Ala Lys Gly Thr Ala Cys Lys Ser Arg Pro Leu Ser Gln Gly
195 200 205
Asp Cys Pro Pro Leu Glu Leu Lys Asn Thr Val Leu Glu Asp Gly Asp
210 215 220
Met Val Asp Thr Gly Tyr Gly Ala Met Asp Phe Ser Thr Leu Gln Asp
225 230 235 240
Thr Lys Cys Glu Val Pro Leu Asp Ile Cys Gln Ser Ile Cys Lys Tyr
245 250 255
Pro Asp Tyr Leu Gln Met Ser Ala Asp Pro Tyr Gly Asp Ser Met Phe
260 265 270
Phe Cys Leu Arg Arg Glu Gln Leu Phe Ala Arg His Phe Trp Asn Arg
275 280 285
Ala Gly Thr Met Gly Asp Thr Val Pro Gln Ser Leu Tyr Ile Lys Gly
290 295 300
Thr Gly Met Arg Ala Ser Pro Gly Ser Cys Val Tyr Ser Pro Ser Pro
305 310 315 320
Ser Gly Ser Ile Val Thr Ser Asp Ser Gln Leu Phe Asn Lys Pro Tyr
325 330 335
Trp Leu His Lys Ala Gln Gly His Asn Asn Gly Val Cys Trp His Asn
340 345 350
Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser Thr Asn Leu Thr
355 360 365
Ile Cys Ala Ser Thr Gln Ser Pro Val Pro Gly Gln Tyr Asp Ala Thr
370 375 380
Lys Phe Lys Gln Tyr Ser Arg His Val Glu Glu Tyr Asp Leu Gln Phe
385 390 395 400
Ile Phe Gln Leu Cys Thr Ile Thr Leu Thr Ala Asp Val Met Ser Tyr
405 410 415
Ile His Ser Met Asn Ser Ser Ile Leu Glu Asp Trp Asn Phe Gly Val
420 425 430
Pro Pro Pro Pro Thr Thr Ser Leu Val Asp Thr Tyr Arg Phe Val Gln
435 440 445
Ser Val Ala Ile Thr Cys Gln Lys Asp Ala Ala Pro Ala Glu Asn Lys
450 455 460
Asp Pro Tyr Asp Lys Leu Lys Phe Trp Asn Val Asp Leu Lys Glu Lys
465 470 475 480
Phe Ser Leu Asp Leu Asp Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val
485 490 495
Gln Ala Gly Leu
500
<210> 19
<211> 524
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(524)
<223> 18L1DE131-138/59dE
<400> 19
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Gly Pro Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys
130 135 140
Pro Ser Asp Val Ile Asn Lys Val Glu Gly Asn Val Ser Glu Asp Val
145 150 155 160
Arg Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu
165 170 175
Gly Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys
180 185 190
Lys Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys
195 200 205
Asn Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala
210 215 220
Met Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp
225 230 235 240
Ile Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala
245 250 255
Asp Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu
260 265 270
Phe Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val
275 280 285
Pro Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly
290 295 300
Ser Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp
305 310 315 320
Ser Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His
325 330 335
Asn Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp
340 345 350
Thr Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro
355 360 365
Val Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His
370 375 380
Val Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr
385 390 395 400
Leu Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile
405 410 415
Leu Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu
420 425 430
Val Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys
435 440 445
Asp Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu Lys Phe
450 455 460
Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp Gln Tyr
465 470 475 480
Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu Arg Arg Lys Pro
485 490 495
Thr Ile Gly Pro Arg Lys Arg Ser Ala Pro Ser Ala Thr Thr Ser Ser
500 505 510
Lys Pro Ala Lys Arg Val Arg Val Arg Ala Arg Lys
515 520
<210> 20
<211> 492
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(492)
<400> 20
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Gly Pro Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys
130 135 140
Pro Ser Asp Val Ile Asn Lys Val Glu Gly Asn Val Ser Glu Asp Val
145 150 155 160
Arg Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu
165 170 175
Gly Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys
180 185 190
Lys Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys
195 200 205
Asn Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala
210 215 220
Met Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp
225 230 235 240
Ile Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala
245 250 255
Asp Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu
260 265 270
Phe Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val
275 280 285
Pro Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly
290 295 300
Ser Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp
305 310 315 320
Ser Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His
325 330 335
Asn Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp
340 345 350
Thr Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro
355 360 365
Val Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His
370 375 380
Val Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr
385 390 395 400
Leu Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile
405 410 415
Leu Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu
420 425 430
Val Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys
435 440 445
Asp Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu Lys Phe
450 455 460
Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp Gln Tyr
465 470 475 480
Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu
485 490
<210> 21
<211> 526
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(526)
<223> 18L1DE121-124/59dE
<400> 21
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Leu Tyr Lys Thr Cys Lys Gln
115 120 125
Ala Gly Thr Cys Pro Ser Asp Val Ile Asn Lys Val Glu Gly Asn Lys
130 135 140
Leu Asp Asp Thr Glu Ser Ser His Ala Ala Thr Ser Asn Val Ser Glu
145 150 155 160
Asp Val Arg Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys
165 170 175
Ile Leu Gly Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr
180 185 190
Ala Cys Lys Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu
195 200 205
Leu Lys Asn Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr
210 215 220
Gly Ala Met Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro
225 230 235 240
Leu Asp Ile Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met
245 250 255
Ser Ala Asp Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu
260 265 270
Gln Leu Phe Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp
275 280 285
Thr Val Pro Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser
290 295 300
Pro Gly Ser Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr
305 310 315 320
Ser Asp Ser Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln
325 330 335
Gly His Asn Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val
340 345 350
Val Asp Thr Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln
355 360 365
Ser Pro Val Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser
370 375 380
Arg His Val Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr
385 390 395 400
Ile Thr Leu Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser
405 410 415
Ser Ile Leu Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr
420 425 430
Ser Leu Val Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys
435 440 445
Gln Lys Asp Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu
450 455 460
Lys Phe Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp
465 470 475 480
Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu Arg Arg
485 490 495
Lys Pro Thr Ile Gly Pro Arg Lys Arg Ser Ala Pro Ser Ala Thr Thr
500 505 510
Ser Ser Lys Pro Ala Lys Arg Val Arg Val Arg Ala Arg Lys
515 520 525
<210> 22
<211> 494
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(494)
<223> 18L1ΔCDE121-124/59dE
<400> 22
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Leu Tyr Lys Thr Cys Lys Gln
115 120 125
Ala Gly Thr Cys Pro Ser Asp Val Ile Asn Lys Val Glu Gly Asn Lys
130 135 140
Leu Asp Asp Thr Glu Ser Ser His Ala Ala Thr Ser Asn Val Ser Glu
145 150 155 160
Asp Val Arg Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys
165 170 175
Ile Leu Gly Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr
180 185 190
Ala Cys Lys Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu
195 200 205
Leu Lys Asn Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr
210 215 220
Gly Ala Met Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro
225 230 235 240
Leu Asp Ile Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met
245 250 255
Ser Ala Asp Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu
260 265 270
Gln Leu Phe Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp
275 280 285
Thr Val Pro Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser
290 295 300
Pro Gly Ser Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr
305 310 315 320
Ser Asp Ser Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln
325 330 335
Gly His Asn Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val
340 345 350
Val Asp Thr Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln
355 360 365
Ser Pro Val Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser
370 375 380
Arg His Val Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr
385 390 395 400
Ile Thr Leu Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser
405 410 415
Ser Ile Leu Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr
420 425 430
Ser Leu Val Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys
435 440 445
Gln Lys Asp Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu
450 455 460
Lys Phe Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp
465 470 475 480
Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu
485 490
<210> 23
<211> 527
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(527)
<223> 18L1h4431-433/59dE
<400> 23
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Asn Val Ser Glu Asp Val Arg
130 135 140
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
145 150 155 160
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
165 170 175
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
180 185 190
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
195 200 205
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
210 215 220
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
225 230 235 240
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
245 250 255
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
260 265 270
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
275 280 285
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
290 295 300
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
305 310 315 320
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
325 330 335
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
340 345 350
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
355 360 365
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
370 375 380
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
385 390 395 400
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
405 410 415
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Leu
420 425 430
Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Ser Asp Val Ile Asn
435 440 445
Lys Val Glu Gly Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys
450 455 460
Leu Lys Phe Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu
465 470 475 480
Asp Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu Arg
485 490 495
Arg Lys Pro Thr Ile Gly Pro Arg Lys Arg Ser Ala Pro Ser Ala Thr
500 505 510
Thr Ser Ser Lys Pro Ala Lys Arg Val Arg Val Arg Ala Arg Lys
515 520 525
<210> 24
<211> 495
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(495)
<223> 18L1ΔCh4431-433/59dE
<400> 24
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Asn Val Ser Glu Asp Val Arg
130 135 140
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
145 150 155 160
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
165 170 175
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
180 185 190
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
195 200 205
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
210 215 220
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
225 230 235 240
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
245 250 255
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
260 265 270
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
275 280 285
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
290 295 300
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
305 310 315 320
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
325 330 335
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
340 345 350
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
355 360 365
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
370 375 380
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
385 390 395 400
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
405 410 415
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Leu
420 425 430
Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Ser Asp Val Ile Asn
435 440 445
Lys Val Glu Gly Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys
450 455 460
Leu Lys Phe Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu
465 470 475 480
Asp Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu
485 490 495
<210> 25
<211> 526
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(526)
<223> 18L1h4432-435/59dE
<400> 25
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Asn Val Ser Glu Asp Val Arg
130 135 140
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
145 150 155 160
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
165 170 175
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
180 185 190
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
195 200 205
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
210 215 220
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
225 230 235 240
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
245 250 255
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
260 265 270
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
275 280 285
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
290 295 300
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
305 310 315 320
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
325 330 335
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
340 345 350
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
355 360 365
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
370 375 380
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
385 390 395 400
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
405 410 415
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp
420 425 430
Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Ser Asp Val Ile
435 440 445
Asn Lys Val Glu Gly Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu
450 455 460
Lys Phe Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp
465 470 475 480
Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu Arg Arg
485 490 495
Lys Pro Thr Ile Gly Pro Arg Lys Arg Ser Ala Pro Ser Ala Thr Thr
500 505 510
Ser Ser Lys Pro Ala Lys Arg Val Arg Val Arg Ala Arg Lys
515 520 525
<210> 26
<211> 494
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(494)
<223> 18L1ΔCh4432-435/59dE
<400> 26
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Asn Val Ser Glu Asp Val Arg
130 135 140
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
145 150 155 160
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
165 170 175
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
180 185 190
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
195 200 205
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
210 215 220
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
225 230 235 240
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
245 250 255
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
260 265 270
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
275 280 285
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
290 295 300
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
305 310 315 320
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
325 330 335
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
340 345 350
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
355 360 365
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
370 375 380
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
385 390 395 400
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
405 410 415
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp
420 425 430
Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Ser Asp Val Ile
435 440 445
Asn Lys Val Glu Gly Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu
450 455 460
Lys Phe Trp Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp
465 470 475 480
Gln Tyr Pro Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu
485 490
<210> 27
<211> 523
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(523)
<223> 18L1DE137-138/59dES-mut1
<400> 27
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Leu Tyr Lys Thr Cys Lys Gln
130 135 140
Ala Gly Thr Cys Pro Ser Asp Val Ile Asn Val Ser Glu Asp Val Arg
145 150 155 160
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
165 170 175
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
180 185 190
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
195 200 205
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
210 215 220
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
225 230 235 240
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
245 250 255
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
260 265 270
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
275 280 285
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
290 295 300
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
305 310 315 320
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
325 330 335
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
340 345 350
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
355 360 365
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
370 375 380
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
385 390 395 400
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
405 410 415
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
420 425 430
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp
435 440 445
Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu Lys Phe Trp
450 455 460
Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp Gln Tyr Pro
465 470 475 480
Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu Arg Gly Gly Pro Thr
485 490 495
Ile Gly Pro Gly Ser Arg Ser Ala Pro Ser Ala Thr Thr Ser Ser Gly
500 505 510
Pro Ala Gly Ser Val Ser Val Gly Ala Gly Lys
515 520
<210> 28
<211> 523
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(523)
<223> 18L1DE137-138/59dES-mut2
<400> 28
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Leu Tyr Lys Thr Cys Lys Gln
130 135 140
Ala Gly Thr Cys Pro Ser Asp Val Ile Asn Val Ser Glu Asp Val Arg
145 150 155 160
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
165 170 175
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
180 185 190
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
195 200 205
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
210 215 220
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
225 230 235 240
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
245 250 255
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
260 265 270
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
275 280 285
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
290 295 300
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
305 310 315 320
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
325 330 335
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
340 345 350
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
355 360 365
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
370 375 380
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
385 390 395 400
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
405 410 415
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
420 425 430
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp
435 440 445
Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu Lys Phe Trp
450 455 460
Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp Gln Tyr Pro
465 470 475 480
Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu Arg Gly Gly Pro Thr
485 490 495
Ile Gly Pro Arg Gly Ser Ser Ala Pro Ser Ala Thr Thr Ser Ser Gly
500 505 510
Pro Ala Gly Ser Val Ser Val Gly Ala Gly Lys
515 520
<210> 29
<211> 523
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(523)
<223> 18L1DE137-138/59dES-mut3
<400> 29
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Leu Tyr Lys Thr Cys Lys Gln
130 135 140
Ala Gly Thr Cys Pro Ser Asp Val Ile Asn Val Ser Glu Asp Val Arg
145 150 155 160
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
165 170 175
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
180 185 190
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
195 200 205
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
210 215 220
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
225 230 235 240
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
245 250 255
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
260 265 270
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
275 280 285
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
290 295 300
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
305 310 315 320
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
325 330 335
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
340 345 350
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
355 360 365
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
370 375 380
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
385 390 395 400
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
405 410 415
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
420 425 430
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp
435 440 445
Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu Lys Phe Trp
450 455 460
Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp Gln Tyr Pro
465 470 475 480
Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu Arg Gly Gly Pro Thr
485 490 495
Ile Gly Pro Gly Ser Arg Ser Ala Pro Ser Ala Thr Thr Ser Ser Gly
500 505 510
Pro Ala Gly Ser Val Gly Val Asp Ala Gly Lys
515 520
<210> 30
<211> 523
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(523)
<223> 18L1DE137-138/59dES-mut4
<400> 30
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Leu Tyr Lys Thr Cys Lys Gln
130 135 140
Ala Gly Thr Cys Pro Ser Asp Val Ile Asn Val Ser Glu Asp Val Arg
145 150 155 160
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
165 170 175
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
180 185 190
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
195 200 205
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
210 215 220
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
225 230 235 240
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
245 250 255
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
260 265 270
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
275 280 285
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
290 295 300
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
305 310 315 320
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
325 330 335
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
340 345 350
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
355 360 365
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
370 375 380
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
385 390 395 400
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
405 410 415
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
420 425 430
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp
435 440 445
Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu Lys Phe Trp
450 455 460
Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp Gln Tyr Pro
465 470 475 480
Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu Arg Gly Gly Pro Thr
485 490 495
Ile Gly Pro Arg Gly Ser Ser Ala Pro Ser Ala Thr Thr Ser Ser Gly
500 505 510
Pro Ala Asp Ser Val Gly Val Asp Ala Gly Lys
515 520
<210> 31
<211> 523
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(523)
<223> 18L1DE137-138/59dES-mut5
<400> 31
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Leu Tyr Lys Thr Cys Lys Gln
130 135 140
Ala Gly Thr Cys Pro Ser Asp Val Ile Asn Val Ser Glu Asp Val Arg
145 150 155 160
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
165 170 175
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
180 185 190
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
195 200 205
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
210 215 220
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
225 230 235 240
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
245 250 255
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
260 265 270
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
275 280 285
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
290 295 300
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
305 310 315 320
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
325 330 335
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
340 345 350
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
355 360 365
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
370 375 380
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
385 390 395 400
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
405 410 415
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
420 425 430
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp
435 440 445
Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu Lys Phe Trp
450 455 460
Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp Gln Tyr Pro
465 470 475 480
Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu Arg Gly Lys Pro Thr
485 490 495
Ile Gly Pro Gly Ser Arg Ser Ala Pro Ser Ala Thr Thr Ser Ser Gly
500 505 510
Pro Ala Gly Ser Val Ser Val Gly Ala Gly Lys
515 520
<210> 32
<211> 523
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(523)
<223> 18L1DE137-138/59dES-mut6
<400> 32
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro
35 40 45
Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Ser Ser His Ala Ala Thr Ser Leu Tyr Lys Thr Cys Lys Gln
130 135 140
Ala Gly Thr Cys Pro Ser Asp Val Ile Asn Val Ser Glu Asp Val Arg
145 150 155 160
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
165 170 175
Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
180 185 190
Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn
195 200 205
Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
210 215 220
Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile
225 230 235 240
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
245 250 255
Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
260 265 270
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro
275 280 285
Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser
290 295 300
Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser
305 310 315 320
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
325 330 335
Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr
340 345 350
Thr Arg Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val
355 360 365
Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val
370 375 380
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
385 390 395 400
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
405 410 415
Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val
420 425 430
Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp
435 440 445
Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu Lys Phe Trp
450 455 460
Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp Gln Tyr Pro
465 470 475 480
Leu Gly Arg Lys Phe Leu Val Gln Ala Gly Leu Arg Gly Lys Pro Thr
485 490 495
Ile Gly Pro Arg Gly Ser Ser Ala Pro Ser Ala Thr Thr Ser Ser Gly
500 505 510
Pro Ala Gly Ser Val Ser Val Gly Ala Gly Lys
515 520
<210> 33
<211> 1477
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> exon
<222> (1)..(1477)
<223> 18L1ΔCDE137-138/59dES nt
<400> 33
atggctctct ggagaccctc cgataacaca gtgtacttgc ccccccccag cgtcgcccgc 60
gtcgtgaaca cagacgacta cgtcaccagg acctcaatct tctaccacgc cggttcaagc 120
cgcctgctga ccgtcggcaa cccctacttc cgcgtccccg ccggtggcgg taacaaacaa 180
gacatcccca aagtcagcgc ctatcagtac cgcgtgttcc gcgtccaact gcccgatccc 240
aacaagttcg gcctgcccga cacctccatc tacaaccccg agacccagag gctggtctgg 300
gcatgcgccg gcgtcgagat cggtaggggc caacccctgg gcgtcggttt gtccggccac 360
cccttctaca acaagctgga cgataccgag tcctcccacg cagcaaccag cctgtacaag 420
acctgcaagc aggccggtac ctgcccctcc gacgtcatca acgtcagcga agatgtccgc 480
gataacgtca gcgtggacta caaacaaacc caactgtgca tcctcggttg cgcacccgcc 540
atcggcgagc attgggccaa gggtaccgcc tgcaagagca ggcccctgag ccaaggtgac 600
tgtccacccc tggagttgaa gaataccgtc ctcgaggacg gcgacatggt ggacaccggc 660
tacggcgcaa tggatttctc caccctgcag gacaccaagt gcgaagtgcc cctcgacatc 720
tgccaaagca tctgcaagta ccccgactac ctgcagatga gcgccgaccc ctacggcgac 780
tccatgttct tctgtctgag aagggaacaa ttgttcgccc gccacttctg gaaccgcgcc 840
ggcaccatgg gcgataccgt cccccagtcc ctgtacatca agggtaccgg catgagggcc 900
agccccggtt catgcgtcta cagcccaagc ccctccggta gcatcgtcac aagcgattcc 960
caactcttca acaagcccta ctggctgcac aaagcccaag gccacaataa cggcgtctgt 1020
tggcacaacc agctgttcgt caccgtcgtg gacacaacca ggtccacaaa cctgaccatc 1080
tgcgccagca cccaaagccc cgtgcccggc cagtacgacg ccacaaagtt caaacaatac 1140
tcacgccacg tcgaagagta cgacctccaa ttcatcttcc aactctgcac catcaccctg 1200
accgccgacg tcatgtccta catccactcc atgaactcat ccatcctgga agactggaat 1260
ttcggcgtcc caccaccccc caccacctcc ctcgtcgaca cctacaggtt cgtgcagagc 1320
gtcgccatca catgccagaa agacgccgcc cccgccgaga acaaagaccc atacgacaaa 1380
ctgaaattct ggaacgtcga cctgaaagag aaattcagcc tggatctgga ccagtaccca 1440
ttgggcagga agttcctcgt ccaggcgggt ctctaat 1477
<210> 34
<211> 1489
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> exon
<222> (1)..(1489)
<223> 18L1ΔCDE137-138/59dE nt
<400> 34
atggctctct ggagaccctc cgataacaca gtgtacttgc ccccccccag cgtcgcccgc 60
gtcgtgaaca cagacgacta cgtcaccagg acctcaatct tctaccacgc cggttcaagc 120
cgcctgctga ccgtcggcaa cccctacttc cgcgtccccg ccggtggcgg taacaaacaa 180
gacatcccca aagtcagcgc ctatcagtac cgcgtgttcc gcgtccaact gcccgatccc 240
aacaagttcg gcctgcccga cacctccatc tacaaccccg agacccagag gctggtctgg 300
gcatgcgccg gcgtcgagat cggtaggggc caacccctgg gcgtcggttt gtccggccac 360
cccttctaca acaagctgga cgataccgag tcctcccacg cagcaaccag cgacctgtac 420
aagacctgca agcaggccgg tacctgcccc tccgacgtca tcaacaaggt caacgtcagc 480
gaagatgtcc gcgataacgt cagcgtggac tacaaacaaa cccaactgtg catcctcggt 540
tgcgcacccg ccatcggcga gcattgggcc aagggtaccg cctgcaagag caggcccctg 600
agccaaggtg actgtccacc cctggagttg aagaataccg tcctcgagga cggcgacatg 660
gtggacaccg gctacggcgc aatggatttc tccaccctgc aggacaccaa gtgcgaagtg 720
cccctcgaca tctgccaaag catctgcaag taccccgact acctgcagat gagcgccgac 780
ccctacggcg actccatgtt cttctgtctg agaagggaac aattgttcgc ccgccacttc 840
tggaaccgcg ccggcaccat gggcgatacc gtcccccagt ccctgtacat caagggtacc 900
ggcatgaggg ccagccccgg ttcatgcgtc tacagcccaa gcccctccgg tagcatcgtc 960
acaagcgatt cccaactctt caacaagccc tactggctgc acaaagccca aggccacaat 1020
aacggcgtct gttggcacaa ccagctgttc gtcaccgtcg tggacacaac caggtccaca 1080
aacctgacca tctgcgccag cacccaaagc cccgtgcccg gccagtacga cgccacaaag 1140
ttcaaacaat actcacgcca cgtcgaagag tacgacctcc aattcatctt ccaactctgc 1200
accatcaccc tgaccgccga cgtcatgtcc tacatccact ccatgaactc atccatcctg 1260
gaagactgga atttcggcgt cccaccaccc cccaccacct ccctcgtcga cacctacagg 1320
ttcgtgcaga gcgtcgccat cacatgccag aaagacgccg cccccgccga gaacaaagac 1380
ccatacgaca aactgaaatt ctggaacgtc gacctgaaag agaaattcag cctggatctg 1440
gaccagtacc cattgggcag gaagttcctc gtccaggcgg gtctctaat 1489
<210> 35
<211> 1486
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> exon
<222> (1)..(1486)
<223> 18L1ΔCDE134-135/59dES nt
<400> 35
atggctctct ggagaccctc cgataacaca gtgtacttgc ccccccccag cgtcgcccgc 60
gtcgtgaaca cagacgacta cgtcaccagg acctcaatct tctaccacgc cggttcaagc 120
cgcctgctga ccgtcggcaa cccctacttc cgcgtccccg ccggtggcgg taacaaacaa 180
gacatcccca aagtcagcgc ctatcagtac cgcgtgttcc gcgtccaact gcccgatccc 240
aacaagttcg gcctgcccga cacctccatc tacaaccccg agacccagag gctggtctgg 300
gcatgcgccg gcgtcgagat cggtaggggc caacccctgg gcgtcggttt gtccggccac 360
cccttctaca acaagctgga cgataccgag tcctcccacg caggaccact gtacaagacc 420
tgcaagcagg ccggtacctg cccctccgac gtcatcccag caaccagcaa cgtcagcgaa 480
gatgtccgcg ataacgtcag cgtggactac aaacaaaccc aactgtgcat cctcggttgc 540
gcacccgcca tcggcgagca ttgggccaag ggtaccgcct gcaagagcag gcccctgagc 600
caaggtgact gtccacccct ggagttgaag aataccgtcc tcgaggacgg cgacatggtg 660
gacaccggct acggcgcaat ggatttctcc accctgcagg acaccaagtg cgaagtgccc 720
ctcgacatct gccaaagcat ctgcaagtac cccgactacc tgcagatgag cgccgacccc 780
tacggcgact ccatgttctt ctgtctgaga agggaacaat tgttcgcccg ccacttctgg 840
aaccgcgccg gcaccatggg cgataccgtc ccccagtccc tgtacatcaa gggtaccggc 900
atgagggcca gccccggttc atgcgtctac agcccaagcc cctccggtag catcgtcaca 960
agcgattccc aactcttcaa caagccctac tggctgcaca aagcccaagg ccacaataac 1020
ggcgtctgtt ggcacaacca gctgttcgtc accgtcgtgg acacaaccag gtccacaaac 1080
ctgaccatct gcgccagcac ccaaagcccc gtgcccggcc agtacgacgc cacaaagttc 1140
aaacaatact cacgccacgt cgaagagtac gacctccaat tcatcttcca actctgcacc 1200
atcaccctga ccgccgacgt catgtcctac atccactcca tgaactcatc catcctggaa 1260
gactggaatt tcggcgtccc accacccccc accacctccc tcgtcgacac ctacaggttc 1320
gtgcagagcg tcgccatcac atgccagaaa gacgccgccc ccgccgagaa caaagaccca 1380
tacgacaaac tgaaattctg gaacgtcgac ctgaaagaga aattcagcct ggatctggac 1440
cagtacccat tgggcaggaa gttcctcgtc caggcgggtc tctaat 1486
<210> 36
<211> 1504
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> exon
<222> (1)..(1504)
<223> 18L1ΔCDE134-135/59dE nt
<400> 36
atggctctct ggagaccctc cgataacaca gtgtacttgc ccccccccag cgtcgcccgc 60
gtcgtgaaca cagacgacta cgtcaccagg acctcaatct tctaccacgc cggttcaagc 120
cgcctgctga ccgtcggcaa cccctacttc cgcgtccccg ccggtggcgg taacaaacaa 180
gacatcccca aagtcagcgc ctatcagtac cgcgtgttcc gcgtccaact gcccgatccc 240
aacaagttcg gcctgcccga cacctccatc tacaaccccg agacccagag gctggtctgg 300
gcatgcgccg gcgtcgagat cggtaggggc caacccctgg gcgtcggttt gtccggccac 360
cccttctaca acaagctgga cgataccgag tcctcccacg caggaccaga cctgtacaag 420
acctgcaagc aggccggtac ctgcccctcc gacgtcatca acaaggtcga aggaccagca 480
accagcaacg tcagcgaaga tgtccgcgat aacgtcagcg tggactacaa acaaacccaa 540
ctgtgcatcc tcggttgcgc acccgccatc ggcgagcatt gggccaaggg taccgcctgc 600
aagagcaggc ccctgagcca aggtgactgt ccacccctgg agttgaagaa taccgtcctc 660
gaggacggcg acatggtgga caccggctac ggcgcaatgg atttctccac cctgcaggac 720
accaagtgcg aagtgcccct cgacatctgc caaagcatct gcaagtaccc cgactacctg 780
cagatgagcg ccgaccccta cggcgactcc atgttcttct gtctgagaag ggaacaattg 840
ttcgcccgcc acttctggaa ccgcgccggc accatgggcg ataccgtccc ccagtccctg 900
tacatcaagg gtaccggcat gagggccagc cccggttcat gcgtctacag cccaagcccc 960
tccggtagca tcgtcacaag cgattcccaa ctcttcaaca agccctactg gctgcacaaa 1020
gcccaaggcc acaataacgg cgtctgttgg cacaaccagc tgttcgtcac cgtcgtggac 1080
acaaccaggt ccacaaacct gaccatctgc gccagcaccc aaagccccgt gcccggccag 1140
tacgacgcca caaagttcaa acaatactca cgccacgtcg aagagtacga cctccaattc 1200
atcttccaac tctgcaccat caccctgacc gccgacgtca tgtcctacat ccactccatg 1260
aactcatcca tcctggaaga ctggaatttc ggtgtcccac caccccccac cacctccctc 1320
gtcgacacct acaggttcgt gcagagcgtc gccatcacat gccagaaaga cgccgccccc 1380
gccgagaaca aagacccata cgacaaactg aaattctgga acgtcgacct gaaagagaaa 1440
ttcagcctgg atctggacca gtacccattg ggcaggaagt tcctcgtcca ggcgggtctc 1500
taat 1504
<210> 37
<211> 1480
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> exon
<222> (1)..(1480)
<223> 18L1ΔCDE131-138/59dE nt
<400> 37
atggctctct ggagaccctc cgataacaca gtgtacttgc ccccccccag cgtcgcccgc 60
gtcgtgaaca cagacgacta cgtcaccagg acctcaatct tctaccacgc cggttcaagc 120
cgcctgctga ccgtcggcaa cccctacttc cgcgtccccg ccggtggcgg taacaaacaa 180
gacatcccca aagtcagcgc ctatcagtac cgcgtgttcc gcgtccaact gcccgatccc 240
aacaagttcg gcctgcccga cacctccatc tacaaccccg agacccagag gctggtctgg 300
gcatgcgccg gcgtcgagat cggtaggggc caacccctgg gcgtcggttt gtccggccac 360
cccttctaca acaagctgga cgataccgag tccggtcccc tgtacaagac ctgcaagcag 420
gccggtacct gcccctccga cgtcatcaac aaggtcgaag gaaacgtcag cgaagatgtc 480
cgcgataacg tcagcgtgga ctacaaacaa acccaactgt gcatcctcgg ttgcgcaccc 540
gccatcggcg agcattgggc caagggtacc gcctgcaaga gcaggcccct gagccaaggt 600
gactgtccac ccctggagtt gaagaatacc gtcctcgagg acggcgacat ggtggacacc 660
ggctacggcg caatggattt ctccaccctg caggacacca agtgcgaagt gcccctcgac 720
atctgccaaa gcatctgcaa gtaccccgac tacctgcaga tgagcgccga cccctacggc 780
gactccatgt tcttctgtct gagaagggaa caattgttcg cccgccactt ctggaaccgc 840
gccggcacca tgggcgatac cgtcccccag tccctgtaca tcaagggtac cggcatgagg 900
gccagccccg gttcatgcgt ctacagccca agcccctccg gtagcatcgt cacaagcgat 960
tcccaactct tcaacaagcc ctactggctg cacaaagccc aaggccacaa taacggcgtc 1020
tgttggcaca accagctgtt cgtcaccgtc gtggacacaa ccaggtccac aaacctgacc 1080
atctgcgcca gcacccaaag ccccgtgccc ggccagtacg acgccacaaa gttcaaacaa 1140
tactcacgcc acgtcgaaga gtacgacctc caattcatct tccaactctg caccatcacc 1200
ctgaccgccg acgtcatgtc ctacatccac tccatgaact catccatcct ggaagactgg 1260
aatttcggcg tcccaccacc ccccaccacc tccctcgtcg acacctacag gttcgtgcag 1320
agcgtcgcca tcacatgcca gaaagacgcc gcccccgccg agaacaaaga cccatacgac 1380
aaactgaaat tctggaacgt cgacctgaaa gagaaattca gcctggatct ggaccagtac 1440
ccattgggca ggaagttcct cgtccaggcg ggtctctaat 1480
<210> 38
<211> 1573
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> exon
<222> (1)..(1573)
<223> 18L1DE137-138/59dES-mut1 nt
<400> 38
atggctctct ggagaccctc cgataacaca gtgtacttgc ccccccccag cgtcgcccgc 60
gtcgtgaaca cagacgacta cgtcaccagg acctcaatct tctaccacgc cggttcaagc 120
cgcctgctga ccgtcggcaa cccctacttc cgcgtccccg ccggtggcgg taacaaacaa 180
gacatcccca aagtcagcgc ctatcagtac cgcgtgttcc gcgtccaact gcccgatccc 240
aacaagttcg gcctgcccga cacctccatc tacaaccccg agacccagag gctggtctgg 300
gcatgcgccg gcgtcgagat cggtaggggc caacccctgg gcgtcggttt gtccggccac 360
cccttctaca acaagctgga cgataccgag tcctcccacg cagcaaccag cctgtacaag 420
acctgcaagc aggccggtac ctgcccctcc gacgtcatca acgtcagcga agatgtccgc 480
gataacgtca gcgtggacta caaacaaacc caactgtgca tcctcggttg cgcacccgcc 540
atcggcgagc attgggccaa gggtaccgcc tgcaagagca ggcccctgag ccaaggtgac 600
tgtccacccc tggagttgaa gaataccgtc ctcgaggacg gcgacatggt ggacaccggc 660
tacggcgcaa tggatttctc caccctgcag gacaccaagt gcgaagtgcc cctcgacatc 720
tgccaaagca tctgcaagta ccccgactac ctgcagatga gcgccgaccc ctacggcgac 780
tccatgttct tctgtctgag aagggaacaa ttgttcgccc gccacttctg gaaccgcgcc 840
ggcaccatgg gcgataccgt cccccagtcc ctgtacatca agggtaccgg catgagggcc 900
agccccggtt catgcgtcta cagcccaagc ccctccggta gcatcgtcac aagcgattcc 960
caactcttca acaagcccta ctggctgcac aaagcccaag gccacaataa cggcgtctgt 1020
tggcacaacc agctgttcgt caccgtcgtg gacacaacca ggtccacaaa cctgaccatc 1080
tgcgccagca cccaaagccc cgtgcccggc cagtacgacg ccacaaagtt caaacaatac 1140
tcacgccacg tcgaagagta cgacctccaa ttcatcttcc aactctgcac catcaccctg 1200
accgccgacg tcatgtccta catccactcc atgaactcat ccatcctgga agactggaat 1260
ttcggcgtcc caccaccccc caccacctcc ctcgtcgaca cctacaggtt cgtgcagagc 1320
gtcgccatca catgccagaa agacgccgcc cccgccgaga acaaagaccc atacgacaaa 1380
ctgaaattct ggaacgtcga cctgaaagag aaattcagcc tggatctgga ccagtaccca 1440
ttgggcagga agttcctcgt ccaggcgggt ctccgtggcg gtccgacgat tggccctggc 1500
tctcgttctg ccccgtcggc cacgaccagc agcggccctg ccggtagcgt gagcgtgggc 1560
gctggcaaat aat 1573
<210> 39
<211> 1573
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> exon
<222> (1)..(1573)
<223> 18L1DE137-138/59dES-mut2 nt
<400> 39
atggctctct ggagaccctc cgataacaca gtgtacttgc ccccccccag cgtcgcccgc 60
gtcgtgaaca cagacgacta cgtcaccagg acctcaatct tctaccacgc cggttcaagc 120
cgcctgctga ccgtcggcaa cccctacttc cgcgtccccg ccggtggcgg taacaaacaa 180
gacatcccca aagtcagcgc ctatcagtac cgcgtgttcc gcgtccaact gcccgatccc 240
aacaagttcg gcctgcccga cacctccatc tacaaccccg agacccagag gctggtctgg 300
gcatgcgccg gcgtcgagat cggtaggggc caacccctgg gcgtcggttt gtccggccac 360
cccttctaca acaagctgga cgataccgag tcctcccacg cagcaaccag cctgtacaag 420
acctgcaagc aggccggtac ctgcccctcc gacgtcatca acgtcagcga agatgtccgc 480
gataacgtca gcgtggacta caaacaaacc caactgtgca tcctcggttg cgcacccgcc 540
atcggcgagc attgggccaa gggtaccgcc tgcaagagca ggcccctgag ccaaggtgac 600
tgtccacccc tggagttgaa gaataccgtc ctcgaggacg gcgacatggt ggacaccggc 660
tacggcgcaa tggatttctc caccctgcag gacaccaagt gcgaagtgcc cctcgacatc 720
tgccaaagca tctgcaagta ccccgactac ctgcagatga gcgccgaccc ctacggcgac 780
tccatgttct tctgtctgag aagggaacaa ttgttcgccc gccacttctg gaaccgcgcc 840
ggcaccatgg gcgataccgt cccccagtcc ctgtacatca agggtaccgg catgagggcc 900
agccccggtt catgcgtcta cagcccaagc ccctccggta gcatcgtcac aagcgattcc 960
caactcttca acaagcccta ctggctgcac aaagcccaag gccacaataa cggcgtctgt 1020
tggcacaacc agctgttcgt caccgtcgtg gacacaacca ggtccacaaa cctgaccatc 1080
tgcgccagca cccaaagccc cgtgcccggc cagtacgacg ccacaaagtt caaacaatac 1140
tcacgccacg tcgaagagta cgacctccaa ttcatcttcc aactctgcac catcaccctg 1200
accgccgacg tcatgtccta catccactcc atgaactcat ccatcctgga agactggaat 1260
ttcggcgtcc caccaccccc caccacctcc ctcgtcgaca cctacaggtt cgtgcagagc 1320
gtcgccatca catgccagaa agacgccgcc cccgccgaga acaaagaccc atacgacaaa 1380
ctgaaattct ggaacgtcga cctgaaagag aaattcagcc tggatctgga ccagtaccca 1440
ttgggcagga agttcctcgt ccaggcgggt ctccgtggcg gtccgacgat tggccctcgt 1500
ggctcttctg ccccgtcggc cacgaccagc agcggccctg ccggtagcgt gagcgtgggc 1560
gctggcaaat aat 1573
<210> 40
<211> 1573
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> exon
<222> (1)..(1573)
<223> 18L1DE137-138/59dES-mut3 nt
<400> 40
atggctctct ggagaccctc cgataacaca gtgtacttgc ccccccccag cgtcgcccgc 60
gtcgtgaaca cagacgacta cgtcaccagg acctcaatct tctaccacgc cggttcaagc 120
cgcctgctga ccgtcggcaa cccctacttc cgcgtccccg ccggtggcgg taacaaacaa 180
gacatcccca aagtcagcgc ctatcagtac cgcgtgttcc gcgtccaact gcccgatccc 240
aacaagttcg gcctgcccga cacctccatc tacaaccccg agacccagag gctggtctgg 300
gcatgcgccg gcgtcgagat cggtaggggc caacccctgg gcgtcggttt gtccggccac 360
cccttctaca acaagctgga cgataccgag tcctcccacg cagcaaccag cctgtacaag 420
acctgcaagc aggccggtac ctgcccctcc gacgtcatca acgtcagcga agatgtccgc 480
gataacgtca gcgtggacta caaacaaacc caactgtgca tcctcggttg cgcacccgcc 540
atcggcgagc attgggccaa gggtaccgcc tgcaagagca ggcccctgag ccaaggtgac 600
tgtccacccc tggagttgaa gaataccgtc ctcgaggacg gcgacatggt ggacaccggc 660
tacggcgcaa tggatttctc caccctgcag gacaccaagt gcgaagtgcc cctcgacatc 720
tgccaaagca tctgcaagta ccccgactac ctgcagatga gcgccgaccc ctacggcgac 780
tccatgttct tctgtctgag aagggaacaa ttgttcgccc gccacttctg gaaccgcgcc 840
ggcaccatgg gcgataccgt cccccagtcc ctgtacatca agggtaccgg catgagggcc 900
agccccggtt catgcgtcta cagcccaagc ccctccggta gcatcgtcac aagcgattcc 960
caactcttca acaagcccta ctggctgcac aaagcccaag gccacaataa cggcgtctgt 1020
tggcacaacc agctgttcgt caccgtcgtg gacacaacca ggtccacaaa cctgaccatc 1080
tgcgccagca cccaaagccc cgtgcccggc cagtacgacg ccacaaagtt caaacaatac 1140
tcacgccacg tcgaagagta cgacctccaa ttcatcttcc aactctgcac catcaccctg 1200
accgccgacg tcatgtccta catccactcc atgaactcat ccatcctgga agactggaat 1260
ttcggcgtcc caccaccccc caccacctcc ctcgtcgaca cctacaggtt cgtgcagagc 1320
gtcgccatca catgccagaa agacgccgcc cccgccgaga acaaagaccc atacgacaaa 1380
ctgaaattct ggaacgtcga cctgaaagag aaattcagcc tggatctgga ccagtaccca 1440
ttgggcagga agttcctcgt ccaggcgggt ctccgtggcg gtccgacgat tggccctggc 1500
tctcgttctg ccccgtcggc cacgaccagc agcggccctg ccggtagcgt gggcgtggac 1560
gctggcaaat aat 1573
<210> 41
<211> 1573
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> exon
<222> (1)..(1573)
<223> 18L1DE137-138/59dES-mut4 nt
<400> 41
atggctctct ggagaccctc cgataacaca gtgtacttgc ccccccccag cgtcgcccgc 60
gtcgtgaaca cagacgacta cgtcaccagg acctcaatct tctaccacgc cggttcaagc 120
cgcctgctga ccgtcggcaa cccctacttc cgcgtccccg ccggtggcgg taacaaacaa 180
gacatcccca aagtcagcgc ctatcagtac cgcgtgttcc gcgtccaact gcccgatccc 240
aacaagttcg gcctgcccga cacctccatc tacaaccccg agacccagag gctggtctgg 300
gcatgcgccg gcgtcgagat cggtaggggc caacccctgg gcgtcggttt gtccggccac 360
cccttctaca acaagctgga cgataccgag tcctcccacg cagcaaccag cctgtacaag 420
acctgcaagc aggccggtac ctgcccctcc gacgtcatca acgtcagcga agatgtccgc 480
gataacgtca gcgtggacta caaacaaacc caactgtgca tcctcggttg cgcacccgcc 540
atcggcgagc attgggccaa gggtaccgcc tgcaagagca ggcccctgag ccaaggtgac 600
tgtccacccc tggagttgaa gaataccgtc ctcgaggacg gcgacatggt ggacaccggc 660
tacggcgcaa tggatttctc caccctgcag gacaccaagt gcgaagtgcc cctcgacatc 720
tgccaaagca tctgcaagta ccccgactac ctgcagatga gcgccgaccc ctacggcgac 780
tccatgttct tctgtctgag aagggaacaa ttgttcgccc gccacttctg gaaccgcgcc 840
ggcaccatgg gcgataccgt cccccagtcc ctgtacatca agggtaccgg catgagggcc 900
agccccggtt catgcgtcta cagcccaagc ccctccggta gcatcgtcac aagcgattcc 960
caactcttca acaagcccta ctggctgcac aaagcccaag gccacaataa cggcgtctgt 1020
tggcacaacc agctgttcgt caccgtcgtg gacacaacca ggtccacaaa cctgaccatc 1080
tgcgccagca cccaaagccc cgtgcccggc cagtacgacg ccacaaagtt caaacaatac 1140
tcacgccacg tcgaagagta cgacctccaa ttcatcttcc aactctgcac catcaccctg 1200
accgccgacg tcatgtccta catccactcc atgaactcat ccatcctgga agactggaat 1260
ttcggcgtcc caccaccccc caccacctcc ctcgtcgaca cctacaggtt cgtgcagagc 1320
gtcgccatca catgccagaa agacgccgcc cccgccgaga acaaagaccc atacgacaaa 1380
ctgaaattct ggaacgtcga cctgaaagag aaattcagcc tggatctgga ccagtaccca 1440
ttgggcagga agttcctcgt ccaggcgggt ctccgtggcg gtccgacgat tggccctcgt 1500
ggctcttctg ccccgtcggc cacgaccagc agcggccctg ccgacagcgt gggcgtggac 1560
gctggcaaat aat 1573
<210> 42
<211> 1573
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> exon
<222> (1)..(1573)
<223> 18L1DE137-138/59dES-mut5 nt
<400> 42
atggctctct ggagaccctc cgataacaca gtgtacttgc ccccccccag cgtcgcccgc 60
gtcgtgaaca cagacgacta cgtcaccagg acctcaatct tctaccacgc cggttcaagc 120
cgcctgctga ccgtcggcaa cccctacttc cgcgtccccg ccggtggcgg taacaaacaa 180
gacatcccca aagtcagcgc ctatcagtac cgcgtgttcc gcgtccaact gcccgatccc 240
aacaagttcg gcctgcccga cacctccatc tacaaccccg agacccagag gctggtctgg 300
gcatgcgccg gcgtcgagat cggtaggggc caacccctgg gcgtcggttt gtccggccac 360
cccttctaca acaagctgga cgataccgag tcctcccacg cagcaaccag cctgtacaag 420
acctgcaagc aggccggtac ctgcccctcc gacgtcatca acgtcagcga agatgtccgc 480
gataacgtca gcgtggacta caaacaaacc caactgtgca tcctcggttg cgcacccgcc 540
atcggcgagc attgggccaa gggtaccgcc tgcaagagca ggcccctgag ccaaggtgac 600
tgtccacccc tggagttgaa gaataccgtc ctcgaggacg gcgacatggt ggacaccggc 660
tacggcgcaa tggatttctc caccctgcag gacaccaagt gcgaagtgcc cctcgacatc 720
tgccaaagca tctgcaagta ccccgactac ctgcagatga gcgccgaccc ctacggcgac 780
tccatgttct tctgtctgag aagggaacaa ttgttcgccc gccacttctg gaaccgcgcc 840
ggcaccatgg gcgataccgt cccccagtcc ctgtacatca agggtaccgg catgagggcc 900
agccccggtt catgcgtcta cagcccaagc ccctccggta gcatcgtcac aagcgattcc 960
caactcttca acaagcccta ctggctgcac aaagcccaag gccacaataa cggcgtctgt 1020
tggcacaacc agctgttcgt caccgtcgtg gacacaacca ggtccacaaa cctgaccatc 1080
tgcgccagca cccaaagccc cgtgcccggc cagtacgacg ccacaaagtt caaacaatac 1140
tcacgccacg tcgaagagta cgacctccaa ttcatcttcc aactctgcac catcaccctg 1200
accgccgacg tcatgtccta catccactcc atgaactcat ccatcctgga agactggaat 1260
ttcggcgtcc caccaccccc caccacctcc ctcgtcgaca cctacaggtt cgtgcagagc 1320
gtcgccatca catgccagaa agacgccgcc cccgccgaga acaaagaccc atacgacaaa 1380
ctgaaattct ggaacgtcga cctgaaagag aaattcagcc tggatctgga ccagtaccca 1440
ttgggcagga agttcctcgt ccaggcgggt ctccgtggca aaccgacgat tggccctggc 1500
tctcgttctg ccccgtcggc cacgaccagc agcggccctg ccggtagcgt gagcgtgggc 1560
gctggcaaat aat 1573
<210> 43
<211> 1573
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> exon
<222> (1)..(1573)
<223> 18L1DE137-138/59dES-mut6 nt
<400> 43
atggctctct ggagaccctc cgataacaca gtgtacttgc ccccccccag cgtcgcccgc 60
gtcgtgaaca cagacgacta cgtcaccagg acctcaatct tctaccacgc cggttcaagc 120
cgcctgctga ccgtcggcaa cccctacttc cgcgtccccg ccggtggcgg taacaaacaa 180
gacatcccca aagtcagcgc ctatcagtac cgcgtgttcc gcgtccaact gcccgatccc 240
aacaagttcg gcctgcccga cacctccatc tacaaccccg agacccagag gctggtctgg 300
gcatgcgccg gcgtcgagat cggtaggggc caacccctgg gcgtcggttt gtccggccac 360
cccttctaca acaagctgga cgataccgag tcctcccacg cagcaaccag cctgtacaag 420
acctgcaagc aggccggtac ctgcccctcc gacgtcatca acgtcagcga agatgtccgc 480
gataacgtca gcgtggacta caaacaaacc caactgtgca tcctcggttg cgcacccgcc 540
atcggcgagc attgggccaa gggtaccgcc tgcaagagca ggcccctgag ccaaggtgac 600
tgtccacccc tggagttgaa gaataccgtc ctcgaggacg gcgacatggt ggacaccggc 660
tacggcgcaa tggatttctc caccctgcag gacaccaagt gcgaagtgcc cctcgacatc 720
tgccaaagca tctgcaagta ccccgactac ctgcagatga gcgccgaccc ctacggcgac 780
tccatgttct tctgtctgag aagggaacaa ttgttcgccc gccacttctg gaaccgcgcc 840
ggcaccatgg gcgataccgt cccccagtcc ctgtacatca agggtaccgg catgagggcc 900
agccccggtt catgcgtcta cagcccaagc ccctccggta gcatcgtcac aagcgattcc 960
caactcttca acaagcccta ctggctgcac aaagcccaag gccacaataa cggcgtctgt 1020
tggcacaacc agctgttcgt caccgtcgtg gacacaacca ggtccacaaa cctgaccatc 1080
tgcgccagca cccaaagccc cgtgcccggc cagtacgacg ccacaaagtt caaacaatac 1140
tcacgccacg tcgaagagta cgacctccaa ttcatcttcc aactctgcac catcaccctg 1200
accgccgacg tcatgtccta catccactcc atgaactcat ccatcctgga agactggaat 1260
ttcggcgtcc caccaccccc caccacctcc ctcgtcgaca cctacaggtt cgtgcagagc 1320
gtcgccatca catgccagaa agacgccgcc cccgccgaga acaaagaccc atacgacaaa 1380
ctgaaattct ggaacgtcga cctgaaagag aaattcagcc tggatctgga ccagtaccca 1440
ttgggcagga agttcctcgt ccaggcgggt ctccgtggca aaccgacgat tggccctcgt 1500
ggctcttctg ccccgtcggc cacgaccagc agcggccctg ccggtagcgt gagcgtgggc 1560
gctggcaaat aat 1573
<210> 44
<211> 23
<212> PRT
<213> HPV 35
<400> 44
Thr Gln Leu Tyr Arg Thr Cys Lys Ala Ala Gly Thr Cys Pro Pro Asp
1 5 10 15
Val Ile Pro Lys Val Glu Gly
20
<210> 45
<211> 23
<212> PRT
<213> HPV 39
<400> 45
Ser Thr Leu Tyr Arg Thr Cys Lys Gln Ser Gly Thr Cys Pro Pro Asp
1 5 10 15
Val Val Asp Lys Val Glu Gly
20
<210> 46
<211> 23
<212> PRT
<213> HPV 51
<400> 46
Thr Gln Leu Tyr Ser Thr Cys Lys Ala Ala Gly Thr Cys Pro Pro Asp
1 5 10 15
Val Val Asn Lys Val Glu Gly
20
<210> 47
<211> 23
<212> PRT
<213> HPV 53
<400> 47
Thr Gln Leu Tyr Gln Thr Cys Lys Gln Ser Gly Thr Cys Pro Glu Asp
1 5 10 15
Val Ile Asn Lys Ile Glu His
20
<210> 48
<211> 23
<212> PRT
<213> HPV 56
<400> 48
Thr Gln Leu Tyr Lys Thr Cys Lys Leu Ser Gly Thr Cys Pro Glu Asp
1 5 10 15
Val Val Asn Lys Ile Glu Gln
20
<210> 49
<211> 23
<212> PRT
<213> HPV 59
<400> 49
Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Ser Asp Val Ile
1 5 10 15
Asn Lys Val Glu Gly Thr Thr
20
<210> 50
<211> 23
<212> PRT
<213> HPV 68
<400> 50
Ser Thr Leu Tyr Lys Thr Cys Lys Gln Ser Gly Thr Cys Pro Pro Asp
1 5 10 15
Val Ile Asn Lys Val Glu Gly
20
<210> 51
<211> 23
<212> PRT
<213> HPV 82
<400> 51
Thr Gln Leu Tyr Ser Thr Cys Lys Ala Ala Gly Thr Cys Pro Pro Asp
1 5 10 15
Val Ile Pro Lys Val Lys Gly
20

Claims (12)

1. A human papillomavirus chimeric protein consisting of HPV type 18L 1 protein or a mutant of HPV type 18L 1 protein and a polypeptide from HPV type 59L 2 protein inserted into the surface region of the HPV type 18L 1 protein or mutant of HPV type 18L 1 protein, wherein:
the amino acid sequence of the HPV18 type L1 protein is shown as SEQ ID NO.1,
the mutant of HPV18 type L1 protein is selected from any one of the following:
a mutant with 32 truncated amino acids at the C end of the amino acid sequence shown in SEQ ID No. 1;
a mutant in which amino acids 477, 478, 485, 496, 502, 506 of the amino acid sequence shown in SEQ ID No.1 are substituted with glycine (G), amino acids 486, 500 are substituted with serine (S), and amino acids 499, 504 are substituted with aspartic acid (D); and
a mutant in which amino acids 477, 484, 496, 499, 504, 506 of the amino acid sequence shown in SEQ ID No.1 are substituted with glycine (G) and amino acids 485, 500, 502 are substituted with serine (S);
the polypeptide from HPV59 type L2 protein is selected from the polypeptide shown in any one of SEQ ID No.4, SEQ ID No.5 or SEQ ID No. 6;
wherein the amino acid sequence of the human papillomavirus chimeric protein is shown in SEQ ID NO: 8. 10, 16, 18, 30 and 31.
2. A polynucleotide encoding the human papillomavirus chimeric protein of claim 1.
3. The polynucleotide of claim 2, wherein the sequence of the polynucleotide is optimized whole gene with e.coli codons or whole gene with insect cell codons.
4. A polynucleotide according to claim 2 or 3 wherein the sequence of the polynucleotide is as set out in any one of SEQ ID No.33, SEQ ID No.34, SEQ ID No.35, SEQ ID No.36, SEQ ID No.41 and SEQ ID No. 42.
5. A vector comprising the polynucleotide of claim 4.
6. A cell comprising the vector of claim 5.
7. A multimer that is a chimeric pentamer or chimeric virus-like particle formed from the human papillomavirus chimeric protein of claim 1.
8. Use of a human papillomavirus chimeric protein according to claim 1 or a multimer according to claim 7 in the preparation of a vaccine for preventing papillomavirus infection and/or a disease induced by papillomavirus infection, wherein for a human papillomavirus chimeric protein as shown in SEQ ID No.31, the papillomavirus is one or more types selected from the group consisting of: HPV18, HPV39, HPV45, HPV59, HPV68, HPV70; for the sequence set forth in SEQ ID NO: 8. 10, 16, 18 and 30, said papillomavirus being one or more types selected from the group consisting of: HPV18, HPV26, HPV33, HPV35, HPV39, HPV45, HPV52, HPV53, HPV59, HPV66, HPV70, HPV73, HPV6, HPV11, HPV2, HPV5 and HPV57.
9. The use according to claim 8, wherein the papillomavirus infection-induced disease is selected from cervical cancer, vaginal cancer, labial cancer, penile cancer, perianal cancer, oropharyngeal cancer, tonsillar cancer, and oral cancer.
10. A vaccine for preventing papillomavirus infection and/or papillomavirus infection-induced disease comprising the human papillomavirus chimeric protein of claim 1 or the multimer of claim 7, and an adjuvant, vaccine excipient, or carrier.
11. Vaccine for preventing papillomavirus infection and/or papillomavirus infection-induced disease according to claim 10 further comprising virus-like particles or chimeric virus-like particles of at least one HPV of the mucophilic and/or dermatological group.
12. Vaccine for use in the prevention of papillomavirus infection and/or papillomavirus infection-induced disease according to claim 10 or 11, wherein the adjuvant is a human adjuvant.
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